CN102573917A

CN102573917A - Pegylated L-asparaginase

Info

Publication number: CN102573917A
Application number: CN2010800303926A
Authority: CN
Inventors: T·阿布里巴
Original assignee: Alize Pharma 2 SAS
Current assignee: Jazz Pharmaceuticals PLC
Priority date: 2009-07-06
Filing date: 2010-07-06
Publication date: 2012-07-11
Anticipated expiration: 2030-07-06
Also published as: US20160060613A1; CO6612174A2; AU2010270294A1; WO2011003886A1; UA104634C2; CA2767149C; ES2636476T5; JP2012532185A; CA2767149A1; US11046946B2; MX2012000424A; DK2451486T3; US9920311B2; ES2636476T3; NZ597912A; HUE035771T2; HK1173092A1; PE20161325A1; PE20120501A1; JP6014956B2

Abstract

Disclosed is a conjugate of a protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol. In particular, the polyethylene glycol has a molecular weight less than or equal to about 5000 Da and the protein is an L-asparaginase from Erwinia. The conjugate of the invention has shown superior properties such as maintenance of a high level of in vitro activity and an unexpected increase in half-life in vivo. Also disclosed are methods of producing the conjugate and use of the conjugate in therapy. In particular, a method is disclosed for use of the conjugate in the treatment of cancer, particularly Acute Lymphoblastic Leukemia (ALL). More specifically, a method is disclosed for use of the conjugate as a second line therapy for patients who have developed hypersensitivity or have had a disease relapse after treatment with other L-asparaginase preparations.

Description

The altheine enzyme of Pegylation

Background of invention

Background technology

Have the active albumen of altheine amidohydrolase (being commonly referred to as the altheine enzyme) and be successfully used to treat the acute lymphoblastic leukemia (ALL) among the child for many years.ALL is modal children malignant tumors (Avramis and Panosyan, Clin.Pharmacokinet. (2005) 44:367-393).

The altheine enzyme also is used to treat Hokdkin disease, acute myeloid leukemia, acute myelomonocyte leukemia; Chronic lymphocytic leukemia; Lymphosarcoma, reticulosarcoma and malignant melanoma (Kotzia and Labrou, J.Biotechnol.127 (2007) 657-669).The anti-tumor activity of altheine enzyme is believed because some malignant cell can not synthesize altheine or reduce the ability (Kotzia and Labrou, J.Biotechnol.127 (2007) 657-669) of synthetic altheine.These malignant cells depend on the extracellular of altheine and supply with.Yet; Altheine enzyme catalysis altheine is hydrolyzed to Radix Asparagi amino acid and ammonia; Thereby exhaust circulation altheine storehouse and kill tumor cell; Said tumor cell can not carry out protein synthesis (Kotzia and Labrou, J.Biotechnol.127 (2007) 657-669) when not having altheine.

From colibacillary altheine enzyme is to be used for first kind of enzyme medicine of ALL treatment and in U.S.'s listing as

, perhaps to go on the market with altheine enzyme

as

in Europe.Also from other microorganism, separated the altheine enzyme; For example; Altheine pheron from chrysanthemum Erwinia (Erwinia chrysanthemi); Be named as Ke Lita enzyme (crisantaspase); It is listing (Wriston Jr., J.C. (1985) " L-asparaGlnase " Meth.Enzymol.113,608-618 as

; Goward, people such as C.R.. (1992) " Rapid large scale preparation of recombinant Erwinia chrysanthemi L-asparaGlnase ", Bioseparation 2,335-341).Also differentiated altheine enzyme from other Erwinia strain; Comprise for example chrysanthemum Erwinia 3937 (Genbank accession number AAS67028); Chrysanthemum Erwinia NCPPB 1125 (Genbank accession number CAA31239), carrot soft rot Erwinia (Erwinia carotovora) (Genbank accession number AAP92666) and carrot soft rot Erwinia Rhizoma Solani tuber osi are deceived shin subspecies (Erwinia carotovora subsp.Astroseptica) (Genbank accession number AAS67027).These chrysanthemum Erwinia altheine enzymes have the amino acid sequence identity of about 91%-98% to each other; And carrot soft rot Erwinia altheine enzyme and chrysanthemum Erwinia altheine enzyme have the amino acid sequence identity (Kotzia and Labrou, J.Biotechnol.127 (2007) 657-669) of about 75%-77%.

The altheine enzyme of bacterial origin has high immunogen and antigen potentiality and often causes untoward reaction; The scope of said untoward reaction is spent the quick anaphylactic shock (Wang that is reacted among the sensitization patient on the lenient side; B. wait the people. (2003) " Evaluation of immunologic cross reaction of anti-asparaGlnase antibodies in acute lymphoblastic leukemia (ALL and lymphoma patients); Leukemia 17,1583-1588).The bacillus coli L-asparaginase enzyme is to have immunogenicly especially, it is reported after i.v. or i.m. use, and has the anti-agedoite enzyme antibody of Chinese People's Anti-Japanese Military and Political College's enterobacteria altheine enzyme: in the adult up to 78%; Be 70% in the child (Wang, people such as B.. (2003) Leukemia 17,1583-1588).

From the altheine enzyme of escherichia coli and chrysanthemum Erwinia different aspect its pharmacokinetic property and have different immunogenicity characteristic spectrum (Klug Albertsen respectively; B. wait the people. (2001) " Comparison of intramuscular therapy with Erwinia asparaGlnase and asparaGlnase Medac:pharmacokinetics; pharmacodynamics; formation of antibodies and influence on thecoagulation system " Brit.J.Haematol.115,983-990).In addition, shown be used for from the altheine enzymes of escherichia coli handle antibody that the back produces not with from the altheine enzyme cross reaction of Erwinia (Wang, people such as B.., Leukemia 17 (2003) 1583-1588).Therefore; From the altheine enzyme of (chrysanthemum) Erwinia in the patient that the bacillus coli L-asparaginase enzyme is responded; Second line treatment (the Duval that is used as ALL; M. wait the people. (2002) " Comparison of Escherichia coli-asparaGlnase with Erwinia-asparaGlnase in the treatment of childhood lymphoid malignancies:results of a randomized European Organisation for Research and Treatment of Cancer; " Blood 15,2734-2739 for Children ' s Leukemia Group phase 3 trial; Avramis and Panosyan, Clin.Pharmacokinet. (2005) 44:367-393).

In order to reduce in the immunogenic trial relevant, produced the bacillus coli L-asparaginase enzyme of modifying through methoxyl group-Polyethylene Glycol (mPEG) at another with the altheine enzyme of using microorganism.The method is commonly referred to as " Pegylation " and has demonstrated and changes proteinic immunogenic properties (Abuchowski; A. wait the people. (1977) " Alteration of Immunological Properties of Bovine Serum Albumin by Covalent Attachment of Polyethylene Glycol; " J.Biol.Chem.252 (11), 3578-3581).This so-called mPEG-L-asparaginase; Or pegaspargase (pegaspargase) is as

(Enzon Inc.; USA) listing; And at first went through as the second line treatment of ALL, and went through from 2006 as the gamma therapy of child and adult's ALL in the U.S. in 1994. has the interior half-life of body of prolongation and the immunogenicity/antigenicity of reduction.

is the bacillus coli L-asparaginase enzyme; Use the mPEG-butanimide succinate (SS-PEG) of 5kDa that it has been carried out modifying (U.S. Patent number 4 at a plurality of lysine residues place; 179,337).SS-PEG is the PEG reagent of the first generation, its contain to the hydrolysis of enzyme responsive, or when the alkalescence pH value unsettled ester bond (U.S. Patent number 4,670,417; Makromol.Chem.1986,187,1131-1144).These character have reduced external and intravital stability and can damage drug safety.

In addition; Shown the Chinese People's Anti-Japanese Military and Political College's enterobacteria altheine enzyme that is produced antibody will with

cross reaction ((Wang; B. wait the people. (2003) " Evaluation of immunologic cross-reaction of anti-asparaGlnase antibodies in acute lymphoblastic leukemia (ALL and lymphoma patients); " Leukemia 17,1583-1588).Though these antibody are not neutralities, this discovery has clearly illustrated that intersection hypersensitivity in vivo or has intersected the high likelihood of inactivation.Really, in a report, the acceptance of 30%-41% the child of pegaspargase have anaphylaxis (Wang, people such as B.. (2003) Leukemia 17,1583-1588).

Except external anaphylaxis, reported the problem of " reticent hypersensitivity " recently, wherein the patient produces anti-agedoite enzyme antibody, but do not demonstrate any allergy clinical indication (Wang, people such as B.. (2003) Leukemia 17,1583-1588).This reaction can cause the neutralizing antibody that forms Chinese People's Anti-Japanese Military and Political College's enterobacteria altheine enzyme and pegaspargase; Yet these patients are not turned to Erwinia altheine enzyme, because there is not external ultra quick sign, therefore, they accept the effective treatment (Holcenberg, J., J.Pediatr.Hematol.Oncol.26 (2004) 273-274) than short-term.

Chrysanthemum Erwinia altheine enzyme treatment is normally used for being derived from the ultra quick situation of colibacillary altheine enzyme.Yet having observed the patient that as many as 30%-50% accepts Erwinia altheine enzyme is antibody positive (Avramis and Panosyan, Clin.Pharmacokinet. (2005) 44:367-393).In addition, because chrysanthemum Erwinia altheine enzyme has the significantly shorter removing half-life than bacillus coli L-asparaginase enzyme, it must be used (Avramis and Panosyan, Clin.Pharmacokinet. (2005) 44:367-393) more frequently.In people's such as Avramis research, the Erwinia asparaginase be associated with the pharmacokinetics characteristic spectrum of difference (people such as Avramis., J.Pediatr.Hematol.Oncol.29 (2007) 239-247).Therefore, with respect to Erwinia altheine enzyme, bacillus coli L-asparaginase enzyme and pegaspargase are the gamma therapies of preferred ALL.

Many bio-pharmaceuticals article are successfully by Pegylation and gone on the market many year.For with PEG and protein coupling, PEG must be activated at its OH end.Based on being selected activated group by obtainable reactive group on the protein of Pegylation.In proteinic situation, most important aminoacid is lysine, cysteine, glutamic acid, aspartic acid, terminal carboxylic acid of C-and N-terminal amino group.In view of reactive group widely in the protein, almost whole chemistry of peptides all is applied to the activated PEG part.The instance of this activatory PEG reagent is activatory carbonic ester, for example, and p-nitrophenyl carbonic ester, succinimdyl carbonate; Active ester, for example, succinimide ester; For locus specificity coupling developed aldehyde and maleimide (Harris, M., Adv.Drug Del.Rev.54 (2002), 459-476).The availability that is used for the various chemical methodes that PEG modifies demonstrate for Pegylation proteic every kind newly developed all will be case study.Except chemical process, the molecular weight that is attached to proteic PEG also has powerful effect for the proteic pharmaceutical properties of Pegylation.In most cases, the molecular weight of expection PEG is high more, the improvement of pharmaceutical properties good more (Sherman, M.R., Adv.Drug Del.Rev.60 (2008), 59-68; Holtsberg, F.W., Journal of Controlled Release 80 (2002), 259-271).For example; People such as Holtsberg find when PEG and arginine deaminase (separating from another microbe-derived amino acid degradation enzyme) when puting together, when the size of PEG attachment is that 5000Da is increased to 20 from molecular weight, during 000Da; The pharmacokinetics and the pharmacodynamic feature of enzyme have increased (Holtsberg; F.W., Journal ofControlled Release 80 (2002), 259-271).

Yet; In many situations; With unmodified the bio-pharmaceuticals article compare; The bio-pharmaceuticals article of Pegylation demonstrate remarkable minimizing activity (Fishburn, C.S. (2008) Review " The Pharmacology of PEGylation:Balancing PD with PK to Generate Novel Therapeutics " J.Pharm.Sci., 1-17).In situation from the altheine enzyme of carrot soft rot Erwinia; Having observed Pegylation makes its external activity be reduced to about 57% (Kuchumova; A.V. wait the people. (2007) " Modification of Recombinant asparaGlnase from Erwinia carotovora with Polyethylene Glycol 5000 " Biochemistry (Moscow) Supplement Series B:Biomedical Chemistry; 1,230-232).Only has about 75% homology from the altheine enzyme of carrot soft rot Erwinia and chrysanthemum Erwinia altheine enzyme (Ke Lita enzyme).Also knownly for

compare with the bacillus coli L-asparaginase enzyme of unmodified, its external activity is about 50%.

The altheine enzyme prepared product that can get does not at present provide such alternative or complementary therapy-particularly treat the therapy of ALL: it is characterized in that high catalytic activity and significantly improved pharmacology and pharmacokinetic properties, and the immunogenicity that reduces.

Invention field

The present invention relates to have the conjugate of active protein of substantial altheine amidohydrolase and Polyethylene Glycol; Particularly wherein said Polyethylene Glycol has the molecular weight of being less than or equal to about 5000Da; Being particularly related to wherein said protein is the conjugate from the altheine enzyme of Erwinia (Erwinia), with and purposes in treatment.

The invention summary

The present invention relates to have the conjugate of active albumen of substantial altheine amidohydrolase and Polyethylene Glycol; Wherein the molecular weight that has of Polyethylene Glycol is less than or equal to about 5000Da, and particularly wherein said protein is the conjugate from the altheine enzyme of Erwinia.In one embodiment; Said conjugate comprises from altheine enzyme of Erwinia and Polyethylene Glycol (PEG); The aminoacid of said altheine enzyme and SEQ ID NO:1 has at least 80% homogeneity, and wherein the molecular weight that has of PEG is less than or equal to about 5000Da.In one embodiment, it is about at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% that the aminoacid of altheine enzyme and SEQ ID NO:1 has, 96%, 97%, 98%, 99% or 100% homogeneity.In some embodiments, the molecular weight that PEG has is about 5000Da, 4000Da, 3000Da, 2500Da or 2000Da.In one embodiment, the altheine enzyme when not puting together with PEG is compared, and the external activity of said conjugate is at least 60%, 65%, and 70%, 75%, 76%, 77%; 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%; 86%, 87%, 88%, 89%, 90%, 91%, 92%; 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.In another embodiment, the altheine enzyme when not puting together with PEG is compared, and the altheine consumption activity that said conjugate has is at least about 10,20, and 30,40,50,60,70,80,90 or 100 times more effective.In another embodiment, said conjugate with blood plasma altheine level be consumed to detection less than level continue at least about 12,24 48,96,108 or 120 hours.

In one embodiment, the altheine enzyme when not puting together with PEG is compared, and said conjugate has the longer body-internal-circulation half-life.In concrete embodiment, said conjugate has longer t than the pegaspargase of using with equal albumen dosage (for example measuring with μ g/kg) (be that PEG puts together, from colibacillary altheine enzyme) _1/2In embodiment more specifically, be that i v uses in the mice after, when the about 50 μ g/kg (based on protein content) of dosage, said conjugate has at least about 58 to about 65 hours t _1/2, and when the about 10 μ g/kg (based on protein content) of dosage, said conjugate has at least about 34 to about 40 hours t _1/2In another embodiment, about 10,000 to about 15,000IU/m ²(about 20-30mg albumen/m ²) dosage range in, said conjugate has at least about 100 to about 200 hours t _1/2In one embodiment, the altheine enzyme when not puting together with PEG is compared, and said conjugate has bigger TG-AUC (AUC).In concrete embodiment, at the albumen dosage that equates, the average A UC that said conjugate has is bigger at least about 3 times than pegaspargase.

In one embodiment, PEG is connected (wherein " amino " comprises that lysine residue and/or N-are terminal) with one or more amino covalences of altheine enzyme.In embodiment more specifically, PEG is connected with one or more amino covalences through amido link.In another concrete embodiment; PEG (for example is covalently attached to about at least 40% to about 100% available amino; Lysine residue and/or proteic N-are terminal) or at least about total amino (for example, lysine residue and/or proteic N-are terminal) of 40% to about 90%.In one embodiment, conjugate has following formula:

Asp-[NH-CO-(CH2)x-CO-NH-PEG]n

Wherein Asp is the altheine enzyme; NH is lysine residue and/or the terminal one or more NH groups of N-of Asp; PEG is a polyalkylene glycol moiety; N is a numeral, and it represent among the Asp the available amino (for example lysine residue and/or N-end) at least about 40% to about 100%, and x is about 1 to about 8, more particularly, about 2 to about 5 integer.In concrete embodiment, PEG is mono methoxy polyethylene glycol (mPEG).

On the other hand, the present invention relates to make the method for conjugate, it is included in and a certain amount of PEG is combined with a certain amount of altheine enzyme be enough to make covalently bound a period of time to the altheine enzyme of PEG.

On the other hand, the present invention relates to pharmaceutical composition, it comprises conjugate of the present invention.

On the other hand, the present invention relates to treat the method for disease, said disease can be treated by consuming the altheine among the patient, and said method comprises the conjugate of the present invention of using effective dose.In one embodiment, said disease is a cancer.In concrete embodiment, said cancer is ALL.In another concrete embodiment, use said conjugate with about 5U/kg body weight to the amount of about 50U/kg body weight.In another embodiment, the dosage range of the conjugate of being used is about 10,000 to about 15,000 IU/m ²(about 20-30mg albumen/m ²).In some embodiments, said using can be intravenous or intramuscular and can be to be less than weekly (for example, every month is once or every other week once), and be weekly, 2 times weekly, or 3 times weekly.In other the specific embodiment, said conjugate is used as monotherapy, and more particularly, does not contain the asparagine synthetase inhibitor.In other embodiment, said conjugate is used (but in some embodiments, said combination treatment does not comprise the asparagine synthetase inhibitor) as the part of combination treatment.In concrete embodiment, the patient who receives treatment has hypersensitivity previously for the form of Colaspase or its Pegylation or for the Erwinia asparaginase.In another embodiment, the patient who receives treatment has palindromia, particularly with the recurrence after the form of therapy of Colaspase or its Pegylation.

Description of drawings

Fig. 1: the SDS-PAGE of purified recombinant chrysanthemum Erwinia altheine enzyme.On SDS-PAGE, analyze the chrysanthemum Erwinia altheine enzyme (r-Ke Lita enzyme) of purified recombinant.With silver nitrate protein band is dyeed.Road 1: molecular weight marker thing (116,66.2,45,35,25,18.4 and 14.4kDa), road 2: purified recombinant chrysanthemum Erwinia altheine enzyme (r-Ke Lita enzyme).

Fig. 2: the SDS-PAGE of mPEG-r-Ke Lita enzyme conjugate analyzes.

Fig. 3:

(5U/kg of dosage in the azygos vein; 25U/kg; 125U/kg and 250U/kg body weight) afterwards, the level of blood plasma altheine.

Fig. 4: in mice behind the single intravenous injection mPEG-r-Ke Lita enzyme conjugate, the blood plasma altheine level of comparing with

.Numeral " 40% " and " 100% " are represented the general degree of the Pegylation of about 40%-55% (the part Pegylation) and about 100% (at utmost Pegylation) available amino respectively.

Fig. 5: in mice behind the single intravenous injection mPEG-r-Ke Lita enzyme conjugate, the TG-AUC (AUC) (residual enzyme is active) that calculates from altheine enzyme characteristic spectrum.

Fig. 6: the following blood plasma altheine level afterwards of dosage in the azygos vein in mice: 2kDa-100%mPEG-r-Ke Lita enzyme (5U/kg; 25U/kg and 50U/kg body weight) (Fig. 6 A); 5kDa-100%mPEG-r-Ke Lita enzyme (5U/kg; 25U/kg and 50U/kg body weight) (Fig. 6 B) or 2kDa-100%mPEG-r-Ke Lita enzyme (5U/kg), 5kDa-100%mPEG-r-Ke Lita enzyme (5U/kg) and pegaspargase

be (Fig. 6 C) (1U/kg).The albumen (10 μ g/kg) of using equivalent produced similar altheine consumption in 72 hours; Said albumen is 2kDa-100%mPEG-r-Ke Lita enzyme (5U/kg); 5kDa-100%mPEG-r-Ke Lita enzyme (5U/kg), or pegaspargase

1U/kg).

Fig. 7: the dose-effect relationship that the r-Ke Lita enzyme of 2kDa-100% Pegylation is compared with the r-Ke Lita enzyme of 5kDa-100% Pegylation.Fig. 7 A shows that with 5U/kg (is 10 μ g/kg based on protein content) when 25U/kg and 50U/kg, after the 2kDa-100% Pegylation r-Ke Lita enzyme of dosage, the residual enzyme in the blood plasma is active in the azygos vein.Fig. 7 B shows that with 5U/kg (is 10 μ g/kg based on protein content) when 25U/kg and 50U/kg, after the r-Ke Lita enzyme of the 5kDa-100% Pegylation of dosage, the residual enzyme in the blood plasma is active in the azygos vein.

Fig. 8: the r-Ke Lita enzyme of 2kDa-100% Pegylation is than the dose-effect relationship of the r-Ke Lita enzyme of 5kDa-100% Pegylation.Behind the 2kDa-100% or 5kDa-100%mPEG-conjugate of the interior dosage of azygos vein, the AUC that the residual enzyme of in mice, measuring is active.Generally speaking, when when identical dosage level compares, the AUC that measures for 5kDa-100%mPEG-r-Ke Lita enzyme is higher than for 2-kDa-100%mPEG-r-Ke Lita enzyme viewed.5,25 when the 50U/kg dosage, observed 31%, 37% and 14% difference respectively.

Fig. 9: in the mice, mPEG-r-Ke Lita enzyme conjugate is with respect to the pharmacokinetics of pegaspargase

.Fig. 9 A represents the 2kDa-100%mPEG-r-Ke Lita enzyme of dosage in the azygos vein; 5kDa-100%mPEG-r-Ke Lita enzyme or pegaspargase

afterwards, the residual enzyme in mice, measured is active.Fig. 9 B represents the 2kDa-100%mPEG-r-Ke Lita enzyme of dosage in the azygos vein; Behind 5kDa-100%mPEG-r-Ke Lita enzyme or the pegaspargase , the AUC that the residual enzyme of in mice, measuring is active.

Figure 10: after mPEG-r-Ke Lita enzyme conjugate or

processing, the serum levels of anti-Ke Lita enzyme spcificity antibody.What antibody was directed against is the Ke Lita enzyme.Data are expressed as meansigma methods ± SD (N=8).

Figure 11: after at utmost the conjugate of (100%) Pegylation is handled with mPEG-r-Ke Lita enzyme, the serum levels of anti-conjugate specific antibody.Figure 11 A: the result is expressed as meansigma methods ± SD (n=8); Figure 11 B: the result is expressed as in anti-conjugate ELISA, has＞percentage rate of the animal of 0.5 absorbance.

Detailed Description Of The Invention

On the one hand, the present invention's problem that will solve provides and has following altheine enzyme prepared product:

The external biological of-Gao is active;

-stable PEG-protein connects;

Half-life in the body of-prolongation;

-the immunogenicity that significantly reduces proves as following: the antibody response that for example reduces or remove anti-altheine enzyme prepared product after the repetitive administration; With

-for the patient who has produced the sensitivity of using a gamma therapy that for example is derived from colibacillary altheine enzyme, as the purposes of two gamma therapies.

Known altheine enzyme conjugate does not address this problem; It has with through the significant cross reactivity of the altheine enzyme prepared product modified (Wang, people such as B.. (2003) Leukemia 17,1583-1588; All incorporate into by reference with it at this); Or have significantly reduced external activity (Kuchumova, people such as A.V.. (2007) Biochemistry (Moscow) Supplement Series B:Biomedical Chemistry, 1; 230-232 all incorporates into it at this by reference).According to the present invention; The purposes of conjugate, the method for preparing this type of conjugate and said conjugate through Erwinia altheine enzyme and hydrophilic polymer (more specifically, molecular weight is 5000Da or Polyethylene Glycol still less) are provided has solved this problem.

What describe among this paper is the altheine enzyme from the Pegylation of Erwinia, compares with the altheine pheron of unmodified and from colibacillary pegaspargase prepared product, and it has improved pharmacological characteristics.The altheine enzyme conjugate of the Pegylation of describing among this paper (for example being the chrysanthemum Erwinia altheine enzyme of the PEG Pegylation of 5000Da by molecular weight) is as therapeutic agent; Be used in particular for being used for from the altheine enzyme of escherichia coli or the altheine enzyme of Pegylation, the treatment of perhaps carrying out from the altheine enzyme of the unmodified of Erwinia shows the patient of excess of export quick (for example anaphylaxis or reticent hypersensitivity).The altheine enzyme conjugate of Pegylation described herein is useful as therapeutics and be used for having palindromia (the for example recurrence of ALL) and the patient that used the asparaginase (for example being used for from the altheine enzyme of escherichia coli or the altheine enzyme of Pegylation) of another kind of form to treat in the past also.

Like what detailed among this paper, to compare with known altheine enzyme (for example pegaspargase) prepared product, conjugate of the present invention demonstrates beat all advantageous characteristic.For example; Compare with altheine enzyme from colibacillary unmodified; Altheine enzyme (Ke Lita enzyme) from the unmodified of chrysanthemum Erwinia has significantly lower half-life (Avramis and Panosyan; Clin.Pharmacokinet. (2005) 44:367-393 all incorporates into it at this by reference).With the protein dosage that equates, the half-life that the conjugate of Pegylation of the present invention has is greater than the altheine enzyme from colibacillary Pegylation.

Definition

Only if definition clearly in addition, term used herein will be understood according to they common implications in the art.

As used herein, term " comprises " and refers to " including but not limited to ", and the term that uses with singulative should comprise plural number, and vice versa, only if context has indication in addition.

As used herein; Term " can through consuming the disease that agedoite is treated " is meant such patient's condition or disease, wherein participates in or facilitates the cell of the said patient's condition or disease to lack the ability of synthetic altheine or have the ability of the synthetic altheine of reduction.The consumption of altheine or forfeiture can be part or (for example, reach use methods known in the art and equipment can not detected level) basically completely.

As used herein, term " treatment effective dose " is meant the amount that produces the needed albumen of desirable therapeutic effect (for example asparaginase or its conjugate).

The altheine pheron

Albumen according to the present invention is to have the active enzyme of altheine amidohydrolase, i.e. altheine enzyme.

Many altheine pherons have been differentiated in the art; It is (to see for example Savitri and Azmi through known method is isolating from microorganism; Indian J.Biotechnol 2 (2003) 184-194 all incorporate into it at this by reference).The most widely-used and the commercial altheine enzyme that gets is derived from escherichia coli or chrysanthemum Erwinia, and the two shares 50% or structural homology still less.In the kind of Erwinia; Being derived from is in the news between the enzyme of chrysanthemum Erwinia and carrot soft rot Erwinia has the sequence of 75%-77% homogeneity usually; And between the different subspecies of chrysanthemum Erwinia, find to have about 90% sequence homogeneity (Kotzia GA; Labrou E, Journal of Biotechnology (2007) 127:657-669 all incorporates into it at this by reference).Some representational Erwinia altheine enzymes comprise those that are for example provided in the table 1:

Table 1

The sequence and the GenBank registration entries of the Erwinia altheine enzyme of table 1 are incorporated at this by reference.Employed preferred altheine enzyme is the altheine enzyme that separates from escherichia coli and Erwinia (particularly chrysanthemum Erwinia) in the treatment.

The altheine enzyme can be the natural enzyme that separates from microorganism.They also can produce in productivity microorganism (for example escherichia coli) through the recombinase technology.As an example; In conjugate of the present invention employed albumen can be in recombination bacillus coli productivity bacterial strain, produce from colibacillary albumen, the albumen of the kind that perhaps in recombination bacillus coli productivity bacterial strain, produces (particularly chrysanthemum Erwinia) from Erwinia.

Can differentiate enzyme through its specific activity.This definition thereby comprise all polypeptide that also are present in the other biological (more particularly, in other microorganism) with defined given activity.Usually can be grouped into some through them and have similar active enzyme as differentiating in PFAM or the defined family of COG.PFAM (the protein families data base of comparison and HMM; Http:// pfam.sanger.ac.uk/) the big set of representing protein sequence to compare.Each PFAM might make that a plurality of comparisons are visual, sees protein domain, estimates in interbiotic distribution, obtains other data base of visit and makes the known protein structures visualization.COG (trooping of proteinic lineal congener group; Http:// www.ncbi.nlm.nih.gov/CQG/) be to obtain through comparing from 43 genomic protein sequences that check order fully, said 43 genomes that check order are fully represented 30 main phylogenys systems.Each COG is definite from least three, the conserved domain before this allows to differentiate.

Differentiate that homologous sequence and their homology and/or the means of homogeneity percentage ratio are well known to those skilled in the art; And particularly including blast program; It can be from the website http://blast.ncbi.nlm.nih.gov/Blast.cgi and being used, use the default parameters that shows on the said website.Can develop the sequence that (for example comparison) obtained then; Use for example program CLUSTALW (http://www.ebi.ac.uk/Tools/clustalw2/index.html) or MULTALIN (http://bioinfo.genotoul.fr/multalin/mutalin.html), the default parameters shown in using on the said website.Use GenBank to go up for the given reference of known gene, those skilled in the art can confirm the gene of the equivalence in other organism, bacterial isolates, yeast, fungus, mammal, the plant etc.The probe that uses consensus sequence advantageously to carry out this conventional work and design degeneracy is cloned corresponding gene in another organism, and said consensus sequence can be confirmed through carrying out sequence alignment with the gene that is derived from other microorganism.These molecular biological conventional methods are well-known to those skilled in the art; And be described in; People (1989MOLECULAR CLONING:A LABORATORY MANUAL.2nd ed.Cold Spring Harbor Lab. such as Sambrook for example; Cold Spring Harbor, New York) in.

Really, it will be understood by those skilled in the art that how to select and design the homologous protein that keeps its altheine enzymatic activity in fact.Usually, use the Nessler algoscopy, according to the method (Mashburn of Mashburn and Wriston description; L.; And Wriston, J. (1963) " Tumor Inhibitory Effect of L-AsparaGlnase, " Biochem Biophys Res Commun 12; 50, all incorporate into by reference with it in this article) confirm the altheine enzymatic activity.

In the specific implementations of conjugate of the present invention, the altheine pheron has homology or homogeneity at least about 80% with the albumen that comprises the sequence of SEQ ID NO:1, and more particularly the albumen with the sequence that comprises SEQ ID NO:1 has at least about 85%, 86% 87%; 88%, 89%, 90%, 91%; 92%, 93%, 94%, 95%; 96%, 97%, 98%, 99% or 100% homology or homogeneity.SEQ ID NO:1 is following:

ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSN?MASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFV?AAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTF?KANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDA?AIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVS?DSLNPAHARILLMLALTRTSDPKVIQEYFHTY(SEQ?ID?NO：1)。

Term " sequence that comprises SEQ ID NO:1 " is meant that proteic aminoacid sequence can strictly not be restricted to SEQ ID NO:1, but can contain other aminoacid.

In specific embodiment, said albumen is the altheine enzyme of chrysanthemum Erwinia, and it has the sequence of SEQ ID NO:1.In another embodiment, the altheine enzyme is from chrysanthemum Erwinia NCPPB 1066 (Genbank accession number CAA32884 all incorporates into it at this by reference), and it has or do not have signal peptide and/or homing sequence.

The proteic fragment of SEQ ID NO:1 also is comprised in the conjugate of the present invention in the employed proteic definition.Term " fragment of SEQ ID NO:1 " is meant that the sequence of polypeptide can comprise the aminoacid still less than SEQ ID NO:1, but still comprises that enough aminoacid gives L-hydrolyzable aminosilane enzymatic activity.

Well known in the artly be, can be through replacing, insert, lack and/or adding one or more aminoacid and come modified polypeptide but keep its enzymatic activity simultaneously.For example, it is common replacing an aminoacid and do not influence proteic functional characteristic through the aminoacid that chemically is equal in given position.Exchange in displacement can be defined as one of following group:

Little aliphatic, nonpolar or polar slightly residue: Ala, Ser, Thr, Pro, Gly

Polarity, electronegative residue and their amide: Asp, Asn, Glu, Gln

The residue of polarity, positively charged: His, Arg, Lys

Big aliphatic, non-polar residue: Met, Leu, Ile, Val, Cys

Big aromatic residue: Phe, Tyr, Trp.

Therefore, can expect that following change will produce the product that is equal on the function: produce with an electronegative residue and replace another change (for example glutamic acid replacement aspartic acid) or produce the change (for example replacing arginine) that replaces another with the residue of a positively charged with lysine.

Adorned position of amino acid residue in the aminoacid sequence and the amino acid whose number that stands to modify are had no particular limits.The technical staff can discern and can be introduced into and do not influence the modification of protein active.For example, can expect that the modification in proteic N or the C-terminal part does not change proteic activity in some cases.Especially, many signs have been carried out, particularly about forming sequence, structure and the residue in active catalytic site about asparaginase.This provides guide about the residue that can not influenced enzymatic activity by modification.All known altheine enzymes from bacterial origin all have common architectural feature.All all is the same tetramer (people such as Aghaipour, Biochemistry 40 (2001) 5655-5664 all incorporate into it at this by reference) that between two adjacent monomeric N and C-terminal domain, has four avtive spots.All all its three grades with quarternary structure in have similarity people such as (, FEBSJ.275 (2008) 4306-4316 all incorporates into it at this by reference) Papageorgiou of height.The sequence of the catalytic site of altheine enzyme is high conservative (people such as Papageorgiou, FEBS is (2008) 4306-4316 J.275) between chrysanthemum Erwinia, carrot soft rot Erwinia and bacillus coli L-asparaginase enzyme II.The avtive spot flexible ring contains amino acid residue 14-33, and structural analysis demonstrates Thr15, Thr95, and Ser62, Glu63, Asp96 contacts part (people such as Papageorgiou, FEBS is (2008) 4306-4316 J.275) with Ala120.People such as Aghaipour have carried out detail analysis people such as (, Biochemistry 40 (2001) 5655-5664) Aghaipour through detecting with the high-resolution crystal structure of the compound enzyme of its substrate to four avtive spots of chrysanthemum Erwinia altheine enzyme.People such as Kotzia provide the sequence from the altheine enzyme of several kinds of Erwinia and subspecies; Though only have the homogeneity of about 75%-77% between the albumen of chrysanthemum Erwinia and carrot soft rot Erwinia; They all still have altheine enzymatic activity (people such as Kotzia separately; J.Biotechnol.127 (2007) 657-669 all incorporates into it at this by reference).People such as Moola have carried out to the epitope mapping research of chrysanthemum Erwinia 3937L-asparaginase and when attempting to reduce the immunogenicity of said asparaginase; Even after the various antigenicity sequences of having suddenlyd change, still can keep enzymatic activity (people such as Moola; Biochem.J.302 (1994) 921-927 all incorporates into it at this by reference).Every piece of article that preceding text are quoted is all incorporated into it at this by reference.In view of the altheine enzyme has been carried out a large amount of signs, those skilled in the art will confirm how to prepare fragment and/or sequence to be replaced and still keeps enzymatic activity.

The polymer that is used for conjugate

Selective polymer from the group of non-toxicity water-soluble polymer, for example polysaccharide (like hetastarch), polyamino acid (like polylysine), polyester (for example polylactic acid), and polyalkylene oxide (like Polyethylene Glycol (PEG)).

Polyethylene Glycol (PEG) or mono methoxy polyethylene glycol (mPEG) be know in this area and comprise linear and ramose polymer.The instance of some polymer (particularly PEG) provides in following, and wherein every piece is all incorporated into it at this by reference: U.S. Patent number 5,672,662; U.S. Patent number 4,179,337; U.S. Patent number 5,252,714; U.S. Patent Application Publication 2003/0114647; U.S. Patent number 6,113,906; U.S. Patent number 7,419,600; With PCT publication number WO2004/083258.

The quality of this base polymer is characterized by polydispersity index (PDI).PDI reflects the distribution of molecular weight in the given polymer samples and is calculated divided by number average molecular weight by weight average molecular weight.The distribution of discrete molecular weight in the polymer of its indication batch.The value of PDI is always greater than 1, but when the approaching desirable Gauss distribution of the chain of polymer (=monodispersity), PDI is near 1.

Polyethylene Glycol advantageously has the molecular weight in the following scope: approximately 500Da is to about 9,000Da.More specifically, Polyethylene Glycol (for example mPEG) has and is selected from following molecular weight: 2000Da, 2500Da, 3000Da, 3500Da, 4000Da, the Polyethylene Glycol of 4500Da and 5000Da.In specific embodiment, the molecular weight that Polyethylene Glycol (for example mPEG) has is 5000Da.

Be used to prepare the method for conjugate

For subsequently with polymer with have the coupling mutually of the active albumen of altheine amidohydrolase, polymer moieties contains activatory functional group, its preferably with albumen in amino reaction.On the one hand, the present invention relates to prepare the method for conjugate, said method comprises makes a certain amount of Polyethylene Glycol (PEG) in solutions buffered, combine to be enough to make PEG and covalently bound a period of time of altheine enzyme with a certain amount of altheine enzyme.In specific embodiment, said altheine enzyme is from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In one embodiment, said PEG is mono methoxy polyethylene glycol (mPEG).

In one embodiment, in the reaction of in solutions buffered, carrying out between Polyethylene Glycol and the altheine enzyme.In some specific embodiments, the pH value of buffer about 7.0 to about 9.0 scope.Most preferred pH value scope is about 7.5 to about 8.5, and for example, pH value is about 7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4 or 8.5.In specific embodiment, the altheine enzyme is from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQID NO:1.

In addition, the Pegylation of altheine enzyme carries out under following protein concentration: about 0.5 to about 25mg/mL, and more particularly approximately 2mg/mL is to about 20mg/mL, and the most about 3mg/mL is 15mg/mL extremely approximately.For example, protein concentration is about 0.5,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20mg/mL.In specific embodiment, the altheine enzyme of Pegylation is from the kind of Erwinia under these protein concentrations, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.

Under the protein concentration of the rising that surpasses 2mg/mL, pegylation reaction carries out fast, is being less than within 2 hours.In addition, applied polymer than the molar excess of the amino in the altheine enzyme for being less than about 20: 1.For example, molar excess is for being less than about 20: 1, and 19: 1,18: 1,17: 1,16: 1,15: 1; 14: 1,13: 1,12: 1,11: 1,10: 1,9: 1,8: 1; 7.5: 1,7: 1,6.5: 1,6: 1,5.5: 1,5: 1,4.5: 1; 4: 1,3.5: 1,3: 1,2.5: 1,2: 1,1.5: 1 or 1: 1.In concrete embodiment, said molar excess is for being less than about 10: 1, and in more concrete embodiment, molar excess is for being less than about 8: 1.In specific embodiment, said altheine enzyme is from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.

Can will depend on the number of free amino group with the number of the peg moiety of albumen coupling, depend on that more which amino can be used for pegylation reaction.In concrete embodiment, the degree of Pegylation (promptly with the altheine enzyme on the number of the mutually link coupled peg moiety of amino) free and/or available amino about 10% to about 100% scope (for example, about 10%, 15%; 20%, 25%, 30%; 40%, 50%, 60%; 70%, 80%, 90% or 100%).100% Pegylation of available amino end (for example, lysine residue and/or proteic N-terminal) also is known as " at utmost Pegylation " in this article.A kind of method of confirming the amino (Pegylation degree) that warp is modified in the mPEG-r-Ke Lita enzyme conjugate is the described method (A.F.S.A.Habeeb of Habeeb; " Determination of free amino groups in proteins by trinitrobenzensulfonic acid "; Anal.Biochem.14 (1966); P.328, all incorporate into by reference with it at this).In one embodiment, the one or more amino coupling mutually of peg moiety and altheine enzyme (wherein amino comprises that lysine residue and/or N-are terminal).In concrete embodiment, the degree of Pegylation total or available amino (for example lysine residue and/or N-are terminal) about 10% to about 100% scope, for example, about 10%, 15%, 20%; 25%, 30%, 35%, 40%, 45%; 50%, 55%, 60%, 65%, 70%; 75%, 80%, 85%, 90%, 95% or 100%.In concrete embodiment, about 40%, 45%, 50%, 55%, 60%, 65%; 70%, 75%, 80%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or 100% total amino (for example, lysine residue and/or N-are terminal) and peg moiety coupling.In another concrete embodiment, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%; 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%; 64%, 65%, 66%, 67%, 68%, 70%, 71%, 72%, 73%, 74%, 75%, 76%; 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% available amino end (for example, lysine residue and/or N-are terminal) and peg moiety coupling.In concrete embodiment, 40%-55% or 100% available amino end (for example, lysine residue and/or N-are terminal) and peg moiety coupling.In some embodiments, peg moiety is through covalently bound and altheine enzyme coupling.In specific embodiment, said altheine enzyme is from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.

In one embodiment, conjugate of the present invention can be expressed from the next:

As?p-[NH-CO-(CH2)x-CO-NH-PEG]n

Wherein Asp is the altheine pheron, and NH is the terminal NH group of lysine residue and/or protein chain N-, and PEG is a polyalkylene glycol moiety, and n is at least 40% to about 100% numeral of available amino end in the albumen (for example lysine residue and/or N-are terminal), and all are all defined in the embodiment of preceding text and hereinafter; X is the integer (for example 1,2,3,4 in 1 to 8 scope; 5,6,7,8); Preferred 2 to 5 (for example 2,3,4,5).In specific embodiment, said altheine enzyme is from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.

Other Pegylation method that can be used for forming conjugate of the present invention is provided in, for example: U.S. Patent number 4,179; 337; U.S. Patent number 5,766,897; Among U.S. Patent Application Publication US 2002/0065397A1 and the U.S. Patent Application Publication US 2009/0054590A1, each piece of writing is wherein all incorporated into it at this by reference.

Concrete embodiment comprises having active albumen of substantial altheine amidohydrolase and Polyethylene Glycol, is selected from the group of conjugate, wherein:

(A)

The altheine enzyme from the chrysanthemum Erwinia shown in-albumen and the SEQ ID NO:1 has at least 90% structural homology,

-Polyethylene Glycol has the molecular weight of about 5000Da,

-albumen and polyalkylene glycol moiety are covalently bound through amido link and albumen, and

-about 100% available amino end (for example, lysine residue and/or N-are terminal) or about 80%-90%, particularly about 84%, total amino (for example, lysine residue and/or N-are terminal) link to each other with polyalkylene glycol moiety.

(B)

The altheine enzyme from the chrysanthemum Erwinia shown in-albumen and the SEQ ID NO:1 has at least 90% homology,

-Polyethylene Glycol has the molecular weight of about 5000Da,

-about 40% to about 45%, particularly about 43% available amino end (for example, lysine residue and/or N-are terminal), or total amino of about 36% (for example, lysine residue and/or N-are terminal) links to each other with polyalkylene glycol moiety.

(C)

-Polyethylene Glycol has the molecular weight of about 2000Da,

-about 100% available amino end (for example, one or more lysine residues and/or N-are terminal) or about 80%-90%, total amino of particularly about 84% (for example, lysine residue and/or N-are terminal) links to each other with polyalkylene glycol moiety.

(D)

-Polyethylene Glycol has the molecular weight of about 2000Da,

-about 50% to about 60%, and particularly about 55% available amino end (for example, lysine residue and/or N-are terminal) or total amino (for example, lysine residue and/or N-are terminal) of about 47% link to each other with polyalkylene glycol moiety.

Altheine enzyme-PEG conjugate

With compare without the altheine enzyme of modifying; Particularly with without the Erwinia altheine enzyme of modifying compare; More particularly with without the altheine enzyme of modifying compare from the chrysanthemum Erwinia; And more particularly compare with the altheine enzyme without modifying of the sequence with SEQ ID NO:1, conjugate of the present invention has some advantage and beat all characteristic.

In some embodiments, when using with the dosage of 5U/kg body weight (bw) or 10 μ g/kg (based on protein content), conjugate of the present invention makes blood plasma altheine level reduce at least about 12,24 48,72,96 or 120 hours time period.In other embodiment, when using with the dosage of 25U/kg bw or 50 μ g/kg (based on protein content), conjugate of the present invention make blood plasma altheine level be reduced to detection less than level continue at least about 12; 24,48,72; 96,120 or 144 hours time period.In other embodiment, when using with the dosage of 50U/kg bw or 100 μ g/kg (based on protein content), conjugate of the present invention reduces at least about 12,24 blood plasma altheine level; 48,72,96,120; 144,168,192,216 or 240 hours time period.In another embodiment, when with about 10,000 to about 15,000IU/m ²(about 20-30mg albumen/m ²) dosage when using, conjugate of the present invention make the level of blood plasma altheine be reduced to detection less than level continue at least about 12,24 48,72,96,120,144,168,192,216 or 240 hours time period.In specific embodiment, said conjugate comprises the altheine enzyme from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In specific embodiment, (molecular weight that for example, mPEG) has is less than or equals about 5000Da the PEG that said conjugate comprises.In more specific embodiment, the available amino end at least about 40% to about 100% (for example, lysine residue and/or N-are terminal) is a Pegylation.

In one embodiment, the monomeric ratio of mol PEG/mol that conjugate comprises is about 4.5 to about 8.5, particularly about 6.5; Specific activity is about 450 to about 550U/mg, particularly about 501U/mg; Compare without the altheine enzyme of modifying with corresponding, relative activity is about 75% to about 85%, particularly about 81%.In concrete embodiment; Conjugate with these characteristics comprises the altheine enzyme from the kind of Erwinia; More particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1; Wherein the available amino end of about 40%-55% (for example, lysine residue and/or N-are terminal) is with the mPEG Pegylation of 5000Da.

In one embodiment, the monomeric ratio of mol PEG/mol that conjugate comprises is about 12.0 to about 18.0, particularly about 15.1; Specific activity is about 450 to about 550U/mg, particularly about 483U/mg; Compare without the altheine enzyme of modifying with corresponding, relative activity is about 75% to about 85%, particularly about 78%.In concrete embodiment; Conjugate with these characteristics comprises the altheine enzyme from the kind of Erwinia; More particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1; Wherein about 100% available amino end (for example, lysine residue and/or N-are terminal) is with the mPEG Pegylation of 5000Da.

In one embodiment, the monomeric ratio of mol PEG/mol that conjugate comprises is about 5.0 to about 9.0, particularly about 7.0; Specific activity is about 450 to about 550U/mg, particularly about 501U/mg; Compare without the altheine enzyme of modifying with corresponding, relative activity is about 80% to about 90%, particularly about 87%.In concrete embodiment; Conjugate with these characteristics comprises the altheine enzyme from the kind of Erwinia, more particularly from the chrysanthemum Erwinia, and more particularly; Said altheine enzyme comprises the sequence of SEQ ID NO:1; Wherein the available amino end of about 40%-55% (for example, lysine residue and/or N-are terminal) is with 10, the mPEG Pegylation of 000Da.

In one embodiment, the monomeric ratio of mol PEG/mol that conjugate comprises is about 11.0 to about 17.0, particularly about 14.1; Specific activity is about 450 to about 550U/mg, particularly about 541U/mg; Compare without the altheine enzyme of modifying with corresponding, relative activity is about 80% to about 90%, particularly about 87%.In concrete embodiment; Conjugate with these characteristics comprises the altheine enzyme from the kind of Erwinia, more particularly from the chrysanthemum Erwinia, and more particularly; Said altheine enzyme comprises the sequence of SEQ ID NO:1; Wherein about 100% available amino end (for example, lysine residue and/or N-are terminal) is with 10, the mPEG Pegylation of 000Da.

In one embodiment, the monomeric ratio of mol PEG/mol that conjugate comprises is about 6.5 to about 10.5, particularly about 8.5; Specific activity is about 450 to about 550U/mg, particularly about 524U/mg; Compare without the altheine enzyme of modifying with corresponding, relative activity is about 80% to about 90%, particularly about 84%.In concrete embodiment; Conjugate with these characteristics comprises the altheine enzyme from the kind of Erwinia; More particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1; Wherein the available amino end of about 40%-55% (for example, lysine residue and/or N-are terminal) is with the mPEG Pegylation of 2000Da.

In one embodiment, the monomeric ratio of mol PEG/mol that conjugate comprises is about 12.5 to about 18.5, particularly about 15.5; Specific activity is about 450 to about 550U/mg, particularly about 515U/mg; Compare without the altheine enzyme of modifying with corresponding, relative activity is about 80% to about 90%, particularly about 83%.In concrete embodiment; Conjugate with these characteristics comprises the altheine enzyme from the kind of Erwinia; More particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1; Wherein about 100% available amino end (for example, lysine residue and/or N-are terminal) is with the mPEG Pegylation of 2000Da.

In other embodiment, behind the single injection, and to compare accordingly without the altheine enzyme of modifying, conjugate of the present invention has at least about 10 times, and 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, the effectiveness of the increase of 90 times or 100 times.In concrete embodiment, the conjugate with these characteristics comprises the altheine enzyme from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In specific embodiment, (molecular weight that for example, mPEG) has is less than or equal to about 5000Da to the PEG that conjugate comprises.In more specific embodiment, the available amino end at least about 40% to about 100% (for example, lysine residue and/or N-are terminal) is a Pegylation.

On the one hand, conjugate of the present invention has the pharmacokinetics characteristic spectrum according to following parameter:

Parameter	Definition
		A _max	Maximum residual enzyme is active
t _Amax	Tester reaches A after exposing _maxTime
		d _Amax	A _maxOr A is at the maximum length in time more than zero

The active half-life time source of residual enzyme is from following formula in the blood plasma:

Average:

t_{1 / 2} = \frac{- In 2 xt}{In (c_{1} / c_{0})}

T wherein _1/2Be the half-life, t is a time point, c _tThe residue blood plasma that is said time point is active, and c ₀Residue blood plasma when being beginning is active.TG-AUC (AUC) is to use pharmacokinetics software (for example, the SigmaPlot version 11) to calculate.

In one embodiment; Conjugate of the present invention has according to following single dose pharmacokinetics characteristic spectrum; Particularly wherein said conjugate comprises that molecular weight is less than or equal to the mPEG of 2000Da and from the altheine enzyme of the kind of Erwinia; More particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1:

A _Max: about 150U/L is to about 250U/L;

T _Amax: about 4h is to about 8h, particularly about 6h;

d _Amax: about 220h is about 250h extremely, especially, and about 238.5h (more than zero, about 90min is to about 240h);

AUC: about 12000 to about 30000; And

t _1/2: about 50h is to about 90h.

In one embodiment; Conjugate of the present invention has according to following single dose pharmacokinetics characteristic spectrum; Particularly wherein said conjugate comprises that molecular weight is less than or equal to the mPEG of 5000Da and from the altheine enzyme of the kind of Erwinia; More particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1:

A _Max: about 18U/L is to about 250U/L;

T _Amax: about 1h is to about 50h;

d _Amax: about 90h is about 250h extremely, about especially 238.5h (more than zero, about 90min is to about 240h);

AUC: about 500 to about 35000; And

t _1/2: about 30h is to about 120h.

In one embodiment, after the single dose, compare the conjugate of the present invention altheine consumption that (for example 24,48 or 72 hours) produce similar level in a period of time with the pegaspargase of equal protein content.In concrete embodiment, conjugate comprises the altheine enzyme from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In specific embodiment, said conjugate comprise have the molecular weight that is less than or equal to about 5000Da PEG (for example, mPEG).In more specific embodiment, be Pegylation at least about 40% available amino end (for example, lysine residue and/or N-are terminal) to about 100% (more particularly, about 40%-55% or 100%).

In one embodiment, compare with the pegaspargase of using with the albumen dosage that equates, conjugate of the present invention has longer t _1/2In concrete embodiment, under the dosage of about 50 μ g/kg (based on protein content), the t that said conjugate has _1/2Be at least about 50,52,54,56,58,59,60,61,62,63,64 or 65 hours.In another concrete embodiment, under the dosage of about 10 μ g/kg (based on protein content), the t that said conjugate has _1/2Be at least about 30,32,34,36,37,38,39 or 40 hours.In another concrete embodiment, about 10,000 to about 15,000IU/m ²(about 20-30mg albumen/m ²) dosage range in, the t that conjugate has _1/2Be at least about 100 to about 200 hours.

In one embodiment, under the albumen dosage that equates, the average A UC that conjugate of the present invention has is bigger at least about 2,3 than pegaspargase, 4 or 5 times.

In one embodiment; After using single dose in one specific period; Conjugate of the present invention does not produce any significant antibody response, and the said time is for for example greater than about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks etc.In specific embodiment, at least 8 weeks of conjugate of the present invention do not produce any significant antibody response.In one embodiment, " do not produce any significant antibody response " and be meant that it is " negative antibody " that the experimenter who accepts said conjugate is differentiated in the parameter of this area approval.Can confirm antibody horizontal through methods known in the art, for example ELISA or surface plasma resonance (SPR-Biacore) measure (people such as Zalewska-Szewczyk., Clin.Exp.Med. (2009) 9:113-116; People such as Avramis., Anticancer Research29 (2009) 299-302, each piece of writing is wherein all incorporated into it at this by reference).Conjugate of the present invention can have the combination in any of these characteristics.

The purposes of Therapeutic Method and conjugate

Conjugate of the present invention can be used for treating can be through consuming the disease that agedoite is treated.For example, said conjugate can be used for treating following or is used for preparing and is used to treat following medicine: adult and child's acute lymphoblastic leukemia (ALL), and expect that the consumption of agedoite will have other patient's condition of useful effect.This type of patient's condition includes but not limited to following: malignant tumor or cancer include but not limited to hematologic malignancies, non-Hodgkin lymphomas, NK lymphoma, cancer of pancreas, lymphogranulomatosis, acute myelocytic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma and melanosarcoma.Comprise the BLOOD DISEASE of immune-mediated in response to the representational non-malignant hematologic disease of agedoite consumption, infectious disease for example is as being infected those (being AIDS) that cause by HIV.The non-BLOOD DISEASE relevant with the agedoite dependency comprises autoimmune disease, for example rheumatoid arthritis, SLE, autoimmune, collagen vascular disease, AIDS etc.Other autoimmune disease comprises osteoarthritis, Ithaca spira's syndrome, psoriasis, insulin-dependent diabetes, multiple sclerosis, causes sclerosis panencephalitis, systemic lupus erythematosus (sle), rheumatic fever, inflammatory bowel (for example, ulcerative colitis and Crohn disease), primary biliary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris and Graves' disease.Can be in any suitable external or body in the algoscopy (for example, wherein growth medium lacks the external test method of agedoite) with regard to the agedoite dependency test suspect the cell that causes disease.Therefore, on the one hand, what the present invention is directed to is the method for medicable disease among the treatment patient, and said method comprises conjugate of the present invention from effective dose to the patient that use.In concrete embodiment, said disease is ALL.In specific embodiment; The conjugate that is used to treat the disease that can treat through agedoite consumption comprises the altheine enzyme from the kind of Erwinia; More particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In specific embodiment, said conjugate comprises the PEG (for example mPEG) with the molecular weight that is less than or equal to about 5000Da.In more specific embodiment, be Pegylation at least about 40% available amino end (for example, lysine residue and/or N-are terminal) to about 100% (more particularly about 40%-55% or 100%).

In one embodiment, the treatment of using conjugate of the present invention to carry out will be used as a gamma therapy.In another embodiment; The treatment of using conjugate of the present invention to carry out will be used in patient (patient that ALL is particularly arranged) as two gamma therapies, and said patient has produced the objective sign of allergy or hypersensitivity (comprising " reticent hypersensitivity ") for other asparaginase prepared product (particularly being derived from the variant (pegaspargase) of colibacillary natural altheine enzyme or its Pegylation).The limiting examples of the objective sign of allergy or hypersensitivity comprises with regard to asparaginase test " antibody positive ".In concrete embodiment, after with the pegaspargase treatment, conjugate of the present invention is used for two gamma therapies.In more specific embodiment, the conjugate that is used for two gamma therapies comprises the altheine enzyme from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In more concrete embodiment, said conjugate also comprises the PEG (for example mPEG) with the molecular weight that is less than or equal to about 5000Da (more particularly, about 5000Da).In addition more concrete embodiment in, be Pegylation at least about 40% available amino end (for example, lysine residue and/or N-are terminal) to about 100% (more particularly, about 40%-55% or 100%).

On the other hand, what the present invention is directed to is the method that is used to treat acute lymphoblastic leukemia, and said method comprises the conjugate of the present invention to patient's administering therapeutic effective dose of the said treatment of needs.In concrete embodiment, will use said treatment with following dosage: about 1500IU/m ²To about 15,000IU/m ², be generally about 10,000 to about 15,000IU/m ²(about 20-30mg albumen/m ²); Scheme for approximately weekly twice to every month once approximately; Be generally once in a week or every other week once; As single reagent (for example monotherapy) or as the part of chemotherapeutic agent combination, said medicine includes but not limited to glucocorticoid, corticosteroid, anticancer compound or other reagent, includes but not limited to methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide and anthracycline antibiotics.As an example, can in 3 chemotherapy phases (comprise and induce, consolidate or strengthen, and keep), use conjugate of the present invention to patient with ALL as the chemotherapeutical component of many reagent.In concrete embodiment, said conjugate is not used with asparagine synthetase inhibitor (for example, those shown in the PCT publication number WO2007/103290 are all incorporated it into it at this by reference) together.In another concrete embodiment, said conjugate is not used with the asparagine synthetase inhibitor, but uses with other chemotherapeutic agent.Said conjugate can be used as the part of many reagent chemotherapy scheme, and before other chemical compound, use simultaneously afterwards or with it.In specific embodiment, said conjugate comprises the altheine enzyme from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In specific embodiment, said conjugate comprise have the molecular weight that is less than or equal to about 5000Da PEG (for example, mPEG).In more specific embodiment kind, be Pegylation at least about 40% available amino end (for example, lysine residue and/or N-are terminal) to about 100% (more particularly, about 40%-55% or 100%).

In concrete embodiment, said method comprises with following amount uses conjugate of the present invention: about 1U/kg is to about 25U/kg (for example about 1,2,3,4,5,6; 7,8,9,10,11,12; 13,14,15,16,17,18; 19,20,21,22,23,24 or 25U/kg) or its equal parts (for example based on protein content).In more concrete embodiment, be selected from down the group amount use said conjugate: about 5, about 10 with about 25U/kg.In another embodiment, use said conjugate with the dosage in the following scope: about 1,000IU/m ²To about 20,000IU/m ²(for example 1,000IU/m ², 2,000IU/m ², 3,000IU/m ², 4,000IU/m ², 5,000IU/m ², 6,000IU/m ², 7,000IU/m ², 8,000IU/m ², 9,000IU/m ², 10,000IU/m ², 11,000IU/m ², 12,000IU/m ², 13,000IU/m ², 14,000IU/m ², 15,000IU/m ², 16,000IU/m ², 17,000IU/m ², 18,000IU/m ², 19,000IU/m ²Or 20,000IU/m ²).In another embodiment, use said conjugate with following dosage: for single dosage, its with altheine be consumed to use method as known in the art and instrument detecting less than lasting about 3 days to about 10 days (for example, 3 of level; 4,5,6; 7,8,9 or 10 days).In another embodiment, said method comprises uses conjugate of the present invention, compares with unconjugated altheine enzyme, and said conjugate causes lower immunogenic response in the patient.In another embodiment, said method comprises uses conjugate of the present invention, compares with unconjugated altheine enzyme, and said conjugate has the longer body-internal-circulation half-life after single dose.In one embodiment, said method comprises uses conjugate of the present invention, and when using with the albumen dosage that equates, said conjugate has the t longer than pegaspargase _1/2In concrete embodiment, said method comprises uses such conjugate, and under the dosage of about 50 μ g/kg (based on protein content), it has at least about 50,52, and 54,56,58,59,60,61,62,63,64 or 65 hours t _1/2In another embodiment, said method comprises uses such conjugate, and under the dosage of about 10 μ g/kg (based on protein content), it has at least about 30,32, and 34,36,37,37,39 or 40 hours t _1/2In another embodiment, said method comprises uses such conjugate, about 10,000 to about 15, and 000IU/m ²(about 20-30mg albumen/m ²) dosage range in, it has at least about 100 to about 200 hours t _1/2In one embodiment, said method comprises uses such conjugate, and under the albumen dosage that equates, its average A UC that has is bigger at least about 2,3 than pegaspargase, 4 or 5 times.In another embodiment, said method comprises uses conjugate of the present invention, and compares without the altheine enzyme of puting together, and it has bigger AUC value after single dose.In specific embodiment, said conjugate comprises the altheine enzyme from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In specific embodiment, said conjugate comprise have the molecular weight that is less than or equal to about 5000Da PEG (for example, mPEG).In more specific embodiment kind, be Pegylation at least about 40% available amino end (for example, lysine residue and/or N-are terminal) to about 100% (more particularly, about 40%-55% or 100%).

After using the altheine enzyme treatment; Relapse rate remains high among the ALL patient; Wherein the department of pediatrics ALL patient of about 10%-25% (for example has early stage recurrence; Some are inducing the keeping of back 30-36 months interim) (Avramis and Panosyan, Clin.Pharmacokinet. (2005) 44:367-393).If the patient through being derived from colibacillary altheine enzyme treatment has recurrence, the treatment of carrying out with the escherichia coli prepared product subsequently can cause " inoculation " effect, and wherein the escherichia coli prepared product has the immunogenicity of increase in application subsequently.In one embodiment; Can conjugate of the present invention be used for treating the patient's of the ALL with recurrence method; Asparaginase prepared product with other before the said patient was treated, and particularly used before those to be derived from the patient that colibacillary agedoite enzyme treatment is crossed.

In some embodiments, purposes of the present invention and Therapeutic Method comprise the altheine enzyme conjugate of using have preceding text (for example, being the part of " altheine enzyme PEG conjugate " at title) or characteristic hereinafter described or property combination.

Compositions, preparation and route of administration

The present invention also comprises the pharmaceutical composition that comprises conjugate of the present invention.In concrete embodiment; Said pharmaceutical composition is comprised in the bottle as freeze dried powder and solvent reconstruct for use; The natural altheine enzyme that for example can get at present; Why in another embodiment for the bacterial origin that no matter is used for its production; Said pharmaceutical composition is the solution of " at any time use ", for example pegaspargase

its make it possible to (after proper process) and use through following: for example intramuscular, intravenous (infuse and/or inject).In brain-ventricle (icv), subcutaneous route.

Can use standard technique to use conjugate of the present invention, comprise the compositions (for example, pharmaceutical composition) that comprises conjugate of the present invention to the patient.Said technology and preparation usually can be at Remington ' s Pharmaceutical Sciences, 18th ed., and Mack Publishing Co., Easton, Pa. found in 1990 (incorporating into by reference at this).

Proper dosage form, a part depend on to be used or route of entry, for example oral, percutaneous, through mucous membrane or through injection (parenteral).This type of dosage form should allow therapeutic agent to arrive target cell, perhaps has required therapeutic effect in addition.For example, the pharmaceutical composition that is injected in the blood flow is preferably soluble.

Can be configured to pharmaceutically acceptable salt and complex thereof according to conjugate of the present invention and/or pharmaceutical composition.Pharmaceutically acceptable salt is atoxic salt when it is existed by amount of being used and concentration.Prepare this type of salt and can not prevent that its its physiologic effect of performance from promoting medicinal application through the physical features that changes chemical compound.Useful change comprises the reduction fusing point with the promotion mucosal administration aspect physical characteristic, and increases dissolubility to promote to use the medicine of higher concentration.The pharmaceutically acceptable salt of asparaginase can be used as complex and exists, and will understand like those skilled in the art.

Pharmaceutically acceptable salt comprises acid-addition salts, for example contains those of sulfate, hydrochlorate, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, mesylate, esilate, benzene sulfonate, tosilate, cyclohexyl-n-sulfonate and quinate.Pharmaceutically acceptable salt can comprise hydrochloric acid, maleic acid, sulphuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethyl sulfonic acid, benzenesulfonic acid, p-methyl benzenesulfonic acid, cyclohexyl sulfamic acid, Fumaric acid and quinic acid available from acid.

Pharmaceutically acceptable salt also comprises base addition salts, for example when existing, contains benzathine benzylpenicillin, chloroprocaine, choline, diethanolamine when acidic functionality (for example carboxylic acid or phenol); Ethylenediamine, meglumine, procaine, aluminum, calcium, lithium; Magnesium, potassium, sodium, ammonium, those of alkylamine and zinc.For example, see Remington ' s Pharmaceutical Sciences, see above.Can use suitable corresponding alkali to prepare this type of salt.

Pharmaceutically acceptable carrier and/or excipient also can be impregnated in according in the pharmaceutical composition of the present invention to promote using of specific asparaginase.The instance that is suitable for the carrier of embodiment of the present invention comprises calcium carbonate, calcium phosphate, and various sugar (for example lactose, glucose or sucrose), or various starch, cellulose derivative, gelatin, vegetable oil, Polyethylene Glycol and physiology go up compatible solvent.The instance of the last compatible solvent of physiology comprises the aseptic aqueous solution (WFI) that is used to inject, saline solution and dextrose.

Can use according to pharmaceutical composition of the present invention through different approach, comprise intravenous, intraperitoneal, subcutaneous, intramuscular, oral, surperficial (percutaneous) or mucosal administration.For general is used, preferably oral.For oral, for example, can chemical compound preparation be become conventional peroral dosage form, for example capsule, tablet and liquid prepared product are like syrup, elixir and concentrated drop.

Alternatively, can use injection (parenteral administration), for example intramuscular, intravenous, intraperitoneal and subcutaneous injection.For injection, pharmaceutical composition is formulated in the liquid solution preferred physiology's compatible buffers or solution, for example saline solution, Hank solution or Ringer solution.In addition, can chemical compound be formulated as solid form and dissolving or suspension again before being about to use.For example, can produce the said conjugate of lyophilized form.In concrete embodiment, intramuscular is used said conjugate.In another embodiment, the said conjugate of intravenous administration.

Also can use through through mucous membrane or percutaneous means realization general.For through mucous membrane or applied dermally, in preparation, use the penetrating agent that is suitable for barrier to be infiltrated.This type of penetrating agent is well known in the art, and for example comprises, for mucosal administration, and cholate and fusidic acid derivatives.In addition, can use detergent to promote infiltration.Mucosal administration for example, can be through nasal spray, inhaler (being used for pulmonary delivery), rectal suppository or vaginal suppository.For surface applied, chemical compound can be configured to ointment, ointment, gel or cream, as known in the art.

The amount of conjugate to be sent will depend on many factors, for example the IC of chemical compound ₅₀, EC ₅₀, biological half life, patient's age, build, weight and physical qualification, and disease to be treated or disease.The importance of these factors and the other factors that will consider is well known to those of ordinary skill in the art.Usually, the scope of the amount of conjugate to be used is about 10 iu/m ²Surface area (the IU/m of patient body ²) to 50,000IU/m ², preferably about 1,000IU/m ²To about 15,000IU/m ²Dosage range, and more preferably about 6,000IU/m ²To about 15,000IU/m ²Scope, for treatment malignant hematologic disease (for example leukemia), particularly preferably be about 10,000 to about 15,000IU/m ²(about 20-30mg albumen/m ²) scope.Usually, in therapeutic process, these dosage be with approximately weekly 3 times to every month interval of (be generally once in a week or every other week once) once approximately, use through intramuscular or intravenous injection.Certainly, also can adopt other dosage and/or therapeutic scheme, as determined by the attending doctor.

In specific embodiment; Conjugate described herein to be used and/or pharmaceutical composition or preparation comprise the altheine enzyme from the kind of Erwinia; More particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In specific embodiment, said conjugate comprises the altheine enzyme from the kind of Erwinia, and more particularly from the chrysanthemum Erwinia, and more particularly, said altheine enzyme comprises the sequence of SEQ ID NO:1.In specific embodiment, said conjugate comprise have the molecular weight that is less than or equal to about 5000Da PEG (for example, mPEG).In more specific embodiment, the amino at least about 40% to about 100% (for example, lysine residue and/or N-are terminal) is Pegylation.

Embodiment

Embodiment 1: the Ke Lita enzyme of preparation reorganization

The recombinant bacteria bacterial strain that is used to make exposed reorganization chrysanthemum Erwinia altheine pheron (being also referred to as " r-Ke Lita enzyme " in this article) is an e. coli strain bl21, and its ansB gene (gene of coding endogenous escherichia coli II type altheine enzyme) with disappearance is to avoid the potentially contaminated of this enzyme for reorganization chrysanthemum Erwinia altheine enzyme.The disappearance of ansB gene depends on the homologous recombination method and according to following three phage transductions that step is carried out:

1) transforms the bacterial isolates (NM 1100) of expressing the defective bacteriophage lambda with linear plasmid (kanamycin box); Wherein said bacteriophage lambda is provided in the bacterial cell protection and the reorganization function through the linear DNA substrate of electroporation, and said linear plasmid contains the kanamycin gene that flank has FLP identification target sequence (FRT).Reorganization taking place to substitute the ansB gene in the bacterial genomes with kanamycin, produces Δ ansB bacterial strain; 2) use the phage transduction will be from the ansB site through kanamycin box Regional Integration to the BL21 bacterial strain integrated of Δ ans B NM1100 bacterial strain.The e. coli strain bl21 that this has produced ansB gene with disappearance and kanamycin has been had resistance; 3) help plasmid to transform this bacterial strain with FLP-to remove kanamycin gene through homologous recombination at FRT sequence place.Genome to final bacterial strain (BL21 Δ ansB bacterial strain) checks order, and confirms the disappearance fully of endogenous ansB gene.

In the DNA sequence insertion expression vector of the escherichia coli-optimization of the ripe chrysanthemum Erwinia altheine enzyme that coding and ENX signal peptide from bacillus subtilis are merged mutually.This carrier allows to induce through adding isopropyl ss-D-1-thio-furan galactoside (IPTG), and under the control of heterozygosis T5/lac promoter express recombinant chrysanthemum Erwinia altheine enzyme and give resistance to kanamycin.

Transform BL21 Δ ansB bacterial strain with this expression vector.To be used to produce r-Ke Lita enzyme through cell transformed through in the Reisenberg culture medium, carrying out the fed-batch glucose fermentation.Cell induce with IPTG as derivant, under 23 ℃, carry out 16h and accomplish.At harvesting and after carrying out cracking through homogenizing in the 10mM sodium phosphate buffer (pH6,5mM EDTA) (buffer A), through with 15000g centrifugal twice and the filtration step of 0.45 μ m and 0.22 μ m subsequently, and the clarification protein solution.Utilize a series of chromatographys and concentration step to come purification of Recombinant chrysanthemum Erwinia altheine enzyme subsequently.In brief, the theoretical isoelectric point, IP (7.23) of chrysanthemum Erwinia altheine enzyme allows recombinase to be adsorbed in cation exchange resin at pH6.Therefore, the enzyme of reorganization is trapped on the Capto S post (cation-exchange chromatography) and with the salt gradient eluting in the buffer A.Compile the fraction that contains recombinase.Then in containing the buffer A of salt gradient, go up the solution that purification is compiled at Capto MMC post (cation-exchange chromatography).Be to carry out before the Protein Separation on the Superdex 200pg size exclusion chromatography, compile the fraction that contains chrysanthemum Erwinia altheine enzyme of institute's eluting and concentrate, as purification step.Compile, concentrate and contain the fraction of recombinase with respect to sodium phosphate buffer (pH8) dialysis of 100mM.Assess the purity of final chrysanthemum Erwinia altheine enzyme prepared product through SDS-PAGE (Fig. 1) and RP-HPLC: it is at least 90%.Integrity through N-end sequencing and LC-MS checking recombinase.Use Nessler ' s reagent to measure enzymatic activity at 37 ℃.The specific activity of purified reorganization chrysanthemum Erwinia altheine enzyme is about 600U/mg.The enzymatic activity of a unit is defined in 37 ℃ of per minutes discharge the enzyme of 1 μ mol ammonia from altheine amount.

Embodiment 2: preparation 10kDa mPEG-L-asparaginase conjugate

In 100mM sodium phosphate buffer (pH 8.0), under 2.5 to 4mg/mL the protein concentration, in the presence of 150mg/mL or 36mg/mL 10kDa mPEG-NHS, in 22 ℃ of stirrings from the solution of the altheine enzyme of chrysanthemum Erwinia 2 hours.Use Superdex 200pg post, in Akta purification appearance UPC 100 systems, the rough 10kDa mPEG-L-asparaginase that comes purification to produce through size exclusion chromatography.Compile and concentrate and contain proteic fraction to produce 2 to 8mg/mL protein concentration.Two kinds of 10kDa mPEG-L-asparaginase conjugates have been prepared by this way; They with without the altheine enzyme of modifying as with reference to, aspect the definite Pegylation degree difference is arranged through the TNBS algoscopy; It is a kind of that (100% available amino end (for example corresponding to complete Pegylation; Lysine residue and/or N-are terminal) residue puted together, the total amino corresponding to 78% (for example, lysine residue and/or N-are terminal) is by Pegylation); Second kind of available amino end (for example, lysine residue and/or N-are terminal)) corresponding to part Pegylation (total amino of 39% (for example, lysine residue and/or N-terminal) or about 50%.SDS-PAGE analysis to conjugate is presented among Fig. 2.The conjugate that is produced is shown as the band of homogeneous basically and does not contain can be detected without the r-Ke Lita enzyme of modifying.

Embodiment 3: preparation 5kDa mPEG-L-asparaginase conjugate

Under the protein concentration in 100mM sodium phosphate buffer (pH 8.0), at 4mg/mL, in the presence of 150mg/mL or 22.5mg/mL 5kDa mPEG-NHS, in 22 ℃ of stirrings from the solution of the altheine enzyme of chrysanthemum Erwinia 2 hours.Use Superdex 200pg post, in Akta purification appearance UPC 100 systems, the rough 5kDa mPEG-L-asparaginase that comes purification to produce through size exclusion chromatography.Compile and concentrate and contain proteic fraction to produce 2 to 8mg/mL protein concentration.Two kinds of 5kDa mPEG-L-asparaginase conjugates have been prepared by this way; Its with without the altheine enzyme of modifying as with reference to, aspect the definite Pegylation degree difference is arranged through the TNBS algoscopy; It is a kind of that (100% available amino end (for example corresponding to complete Pegylation; Lysine residue and/or N-are terminal) residue puted together, the total amino corresponding to 84% (for example, lysine residue and/or N-are terminal) is by Pegylation); Second kind of available amino end (for example, lysine residue and/or N-are terminal)) corresponding to part Pegylation (total amino of 36% (for example, lysine residue and/or N-terminal) or about 43%.SDS-PAGE analysis to conjugate is presented among Fig. 2.The conjugate that is produced is shown as the band of homogeneous basically and does not contain can be detected without the r-Ke Lita enzyme of modifying.

Embodiment 4: preparation 2kDa mPEG-L-asparaginase conjugate

Under the protein concentration in 100mM sodium phosphate buffer (pH 8.0), at 4mg/mL, in the presence of 150mg/mL or 22.5mg/mL 2kDa mPEG-NHS, in 22 ℃ of stirrings from the solution of the altheine enzyme of chrysanthemum Erwinia 2 hours.Use Superdex 200pg post, in Akta purification appearance UPC 100 systems, the rough 2kDa mPEG-L-asparaginase that comes purification to produce through size exclusion chromatography.Compile and concentrate and contain proteic fraction to produce 2 to 8mg/mL protein concentration.Two kinds of 2kDa mPEG-L-asparaginase conjugates have been prepared by this way; Its with without the altheine enzyme of modifying as with reference to, aspect the definite Pegylation degree difference is arranged through the TNBS algoscopy; It is a kind of that (100% available amino end (for example corresponding to Pegylation at utmost; Lysine residue and/or N-are terminal) residue puted together, the total amino corresponding to 86% (for example, lysine residue and/or N-are terminal) is by Pegylation); Second kind of available amino end (for example, lysine residue and/or N-are terminal)) corresponding to part Pegylation (total amino of 47% (for example, lysine residue and/or N-terminal) or about 55%.SDS-PAGE analysis to conjugate is presented among Fig. 2.The conjugate that is produced is shown as the band of homogeneous basically and does not contain can be detected without the r-Ke Lita enzyme of modifying.

The activity of embodiment 5:mPEG-r-Ke Lita enzyme conjugate

Confirm the altheine enzyme hydrolyzable aminosilane enzymatic activity of every kind of conjugate described in the previous embodiment by the Nesslerization of the ammonia that discharges from altheine through enzymatic activity.In brief, the 20mML-agedoite in 50 μ L enzymatic solution and the 50mM sodium borate buffer liquid (pH 8.6) is mixed mutually be incorporated in 37 ℃ of following incubation 10min.The stopped reaction through the Nessler reagent that adds 200 μ L.Measure this solution absorbency at the 450nm place.Calculate said activity by calibration curve, said calibration curve is obtained as reference by ammonium sulfate.The result is summarised in the following table 2:

The activity of table 2:mPEG-r-Ke Lita enzyme conjugate

* numeral " 40% " and " 100% " expression is respectively the roughly Pegylation degree (see embodiment 2-4, see above) of 40%-55% and 100% available amino end.

The mol PEG/mol monomer ratio that * utilizes the data of TNBS algoscopy to know by inference supposes that from proteic all amino (for example, lysine residue and N-are terminal) all be available.

The residual activity of mPEG-r-Ke Lita enzyme conjugate 483 to the scope of 543 units/mg.This is corresponding to the active 78%-87% of altheine amidohydrolase without the enzyme of modifying.

Embodiment 6: without the altheine-drain effect of the Ke Lita enzyme of modifying

The pharmacodynamic properties spectrum of in B6D2F1-heterozygote (immunocompetent, female) (Charles River Germany), having measured

.

is the commercial Ke Lita enzyme that gets (being derived from the altheine enzyme of chrysanthemum Erwinia).In brief; That 2 animal/windings have received is single, 5; 25,125 or the i.v. of 250 units/kg bw injection.6h, 12h, 24h and 48h after 1h and the administration before administration collect plasma sample and with regard to the said blood plasma of the horizontal analysis of altheine from the eye hole.

Measure the blood plasma amino acid levels with PICO-TAG amino acid analysis test kit (Waters).In brief, make the plasma sample Deproteinizationization through methanol extraction.With phenyl isothiocyanate derive in the supernatant free amino acid and carry out quantitatively through RP-HPLC.

As shown in Figure 3, after iv used, 5 did not consume the altheine level in the mice effectively with the dosage of 25U/kg.Have only the dosage of 250U/kg in 48 hours, to induce consumption completely.

The clinical limitation that this result demonstrates

(without the Ke Lita enzyme of modifying); It need be until 3 times/week, suffer from as being injected at of pain among the patient of ALL and used; And use with high dose, this causes common anaphylaxis and immunogenicity.

Embodiment 7: altheine-drain effect and blood plasma altheine enzymatic activity behind six kinds of mPEG-r-Ke Lita of single administration enzyme conjugate

The drug effect and the pharmacokinetics characteristic spectrum of 6 kinds of different mPEG-r-Ke Lita enzyme conjugates in B6D2F1-heterozygote (immunocompetent, magnetic) (Charles River Germany), have been measured.Six kinds of conjugates being tested have difference at the molecular size of PEG (2,5 or 10kDa) and degree (maximum or the part Pegylation) aspect of Pegylation.Use without the Ke Lita enzyme of modifying

as reference.In brief, 4 animal/windings are injected by the i.v. of single 5 units/kg bw conjugate or 250 units/kg bw

.6h, 24h, 48h, 96h and 192h after 1h and the administration before administration, from the eye hole of every animal collect plasma sample and respectively with regard to the blood plasma level of altheine and the residual enzyme activity analyze.

Confirm the enzymatic activity in the blood plasma through the chromogenic assay method.Use L-aspartoyl β-hydroxamic acid (AHA) as substrate.Enzyme is hydrolyzed to L-Asp and azanol with AHA, after carrying out condensation with oxine and being oxidized to indooxine, at 710nm it is measured.(Analytical Biochemistry 309 (2002): 117-126, all incorporate into by reference with it at this).

As shown in Figure 4; It is good equally with

250U/kg at least that the altheine consumption that the conjugate of using with 5U/kg demonstrates is renderd a service, and the expression Pegylation makes at least 50 times of proteic effectiveness increases.All conjugates all demonstrate similar effectiveness, make the blood plasma level of altheine consume 2 days, and (it shows longer acting duration (96h=4 days, than 48h=2 days of other conjugate) except the 5kDa-100% conjugate.

Therefore, will increase to 5kDa from 2kDa with the PEG size that r-Ke Lita enzyme is puted together and produce the effectiveness and the acting duration that improve.Yet, surprisingly, the size of PEG is increased to effectiveness and the acting duration that 10kDa does not further improve conjugate, with 5kDa at utmost the conjugate of Pegylation compare, itself in addition caused decline.

Enzymatic activity is consistent with altheine consumption.As shown in Figure 5, the 5kDa-100% conjugate demonstrates maximum AUC, reflects the longer half-life.With respect to PEG-100% (at utmost Pegylation) conjugate, observed littler AUC and do not observed difference for the 10kDa material standed for for the conjugate with PEG-40% (part Pegylation) of 2kDa and 5kDa candidate.

Consistent with the altheine consumption data, will increase to 5kDa from 2kDa with the PEG molecular size that r-Ke Lita enzyme is puted together and produce longer circulation altheine enzymatic activity.Yet, surprisingly, the size of PEG is increased to enzymatic activity in the body that 10kDa further do not improve conjugate, with 5kDa at utmost the conjugate of Pegylation compare, itself in addition caused decline.In addition, notably, when making r-Ke Lita enzyme with HMW (being 40kDa) mPEG, enzyme there is not appreciable impact (data not shown) for Proteolytic vitro stability at the single Pegylation of N-end.

8: two kinds of mPEG-r-Ke Lita of embodiment enzyme conjugate is for the dosage range effect of blood plasma altheine

2 kinds of pharmacodynamic properties spectrums that mPEG-r-Ke Lita enzyme conjugate is compared with pegaspargase

in B6D2F1-heterozygote (immunocompetent, female) (Charles River Germany), have been measured.The conjugate of being tested be 3 dosage, 2kDa r-Ke Lita enzyme and the 5kDa r-Ke Lita enzyme of (100%) Pegylation at utmost of (100%) Pegylation at utmost.In brief, that 8 animal/windings have received is single, 5,25 or the i.v. injection of the r-Ke Lita enzyme conjugate of 50 units/kg bw, corresponding to 10,50 or 100 μ g albumen/kg.As comparing; With 1 unit/kg (corresponding to 10 μ g albumen/kg) tested

90min, 6h, 24h, 48h, 72h, 96h, 120h after 1h and the administration before administration; 144h; 192h and 240h analyze from eye hole collection plasma sample and with regard to the blood plasma level of altheine.

Conjugate is presented among Fig. 6 for the dosage relevant effect of blood plasma altheine level.Shown in Fig. 6 A and 6B, two kinds of conjugates all are being highly effective aspect the consumption circulation altheine.For 2kDa 100% conjugate, respectively with 5U, the dosage of 25U and 50U/kg, observed 3,6 and at least 10 days in wastage in bulk or weight.For 5kDa 100% conjugate, respectively with 5U, the dosage of 25U and 50U/kg has been observed the wastage in bulk or weight in 3,10 and 10 days.For two kinds of conjugates of being tested, the dosage of 5,25 and the 50U/kg that is tested is compared with the altheine enzyme prepared product that other can get corresponding to 10,50 and 100 μ g/kg (based on protein content), and this is low-down protein content.Really; 250U/kg

is corresponding to about 520 μ g/kg, and 1U/kg

is roughly corresponding to 10 μ g/kg (based on protein content).Fig. 6 C demonstrates 2kDa-100% conjugate, 5kDa-100% conjugate or albumen of using equivalent (10 μ g/kg) and in 72hr, produces similar altheine consumption.

The pharmacokinetics characteristic spectrum of 9: two kinds of mPEG-r-Ke Lita of embodiment enzyme conjugate

In B6D2F1-heterozygote (immunocompetent, female) (Charles River Germany), measured the pharmacokinetics characteristic spectrum of mPEG-r-Ke Lita enzyme conjugate.The conjugate of being tested be 3 dosage, 2kDa r-Ke Lita enzyme and 5kDa (100%) r-Ke Lita enzyme of Pegylation fully at utmost of (100%) Pegylation at utmost.Also tested without the Ke Lita enzyme of modifying

; (250U/kg) with

; (1U/kg) as contrast.In brief; That 8 animal/windings have received is single, 5,25 or the i.v. injection (comparing) of every kind of mPEG-r-Ke Lita enzyme conjugate of 50 units/kg bw with and .90min, 6h, 24h, 48h, 72h, 96h, 120h after 1h and the administration before administration, 144h, 192h and 240h collect plasma sample and analyze with regard to the active blood plasma level of residual enzyme from the eye hole.

Confirm the enzymatic activity in the blood plasma through the chromogenic assay method.Use L-aspartoyl β-hydroxamic acid (AHA) as substrate.Enzyme is hydrolyzed to L-Asp and azanol with AHA, after carrying out condensation with oxine and being oxidized to indooxine, at 710nm it is measured.(Analytical?Biochemistry?309(2002)：117-126)。

In order to calculate time half-life, utilize the MS-excel instrument to obtain the active index line of best fit of each residue blood plasma.From calculate, got rid of minus activity value.

Parameter	Definition
		A _max	Maximum residual enzyme is active
t _Amax	Tester reaches A after exposing _maxTime
		d _Amax	?A _maxOr A is at the maximum length in time more than zero

The half-life time source of residual enzyme activity is from following formula in the blood plasma, and it utilizes the formula separately of the MS-excel instrument and the index line of best fit:

Average:

t_{1 / 2} = \frac{- In 2 xt}{In (c_{1} / c_{0})}

T wherein _1/2Be the half-life, t is a time point, c _tResidue blood plasma when said time point is active, c ₀Residue blood plasma when being beginning is active.TG-AUC (AUC) is to use SigmaPlot version 11 to calculate.Pharmacokinetic data be summarised in following table 3 with 4 and Fig. 7-9 in.

Table 3: 250U/kg? Bw of

1U/kg? bw pegaspargase

or 2kDa? mPEG-r-g and made him 100% of the enzyme conjugate for single processing basic pharmacokinetic (residual plasma activity)

Table 4: 250U/kg? Bw of

1U/kg? bw pegaspargase

or 5kDa? mPEG-r-g and made him 100% of the enzyme conjugate for single processing basic pharmacokinetic (residual plasma activity)

Data show goes out and compares without the Ke Lita enzyme of modifying, the Pegylation significant prolongation of r-Ke Lita enzyme the half-life, and be mode (table 3 and 4, Fig. 7-9) with dose dependent.In addition, when when identical dosage level compares, be higher than for the 2-kDa-100% conjugate viewed for the measured AUC of 5kDa-100%.Find that respectively for the 5kDa-100% conjugate, concordance is found 21%, 37% and 14% difference (Fig. 8) 5,25 with during 50U/kg dosage.When testing based on the same dose of protein content; The 5kDa-100% conjugate also demonstrates to has than

longer half-life itself, shown in the pharmacokinetic parameter that is drawn that shows among Fig. 9 and the table 4.The superior pharmacokinetics characteristic spectrum of Erwinia conjugate is surprising, because knownly be derived from colibacillary altheine enzyme and in humans and animals, have the longer half-life than the altheine enzyme that is derived from the chrysanthemum Erwinia (Ke Lita enzyme).Therefore, the bacillus coli L-asparaginase enzyme (pegaspargase) of logically predicting Pegylation is compared with the r-Ke Lita enzyme of Pegylation to have the longer half-life.Yet, unexpectedly and advantageously, the r-Ke Lita enzyme of Pegylation has the longer half-life than pegaspargase.

Following table 5 has been summed up the pharmacokinetics and the drug effect data of collecting from several experiments; Comprise described in the embodiment 7-9 of this paper those; Its demonstration: 1) 2kDa-100% and 5kDa-100% conjugate all are being highly effective aspect the acting duration of increase rendeing a service with the Ke Lita enzyme, like what viewed significant difference was shown during comparison with ; 2) the 5kDa-100% conjugate all has more secular effect than 2kDa-100% conjugate and , and observed longer the half-life showed like the dosage place of testing to some extent in institute.In view of the poor outcome surprisingly that is obtained with the 10kDa-100% conjugate, the benefit of these data declaration Pegylations increases along with the size that anchors to the peg moiety on the Ke Lita enzyme, until 5kDa.More high-molecular weight PEG does not increase more benefit, and at least in the situation of 10kDa, itself in addition possibly be deleterious.This be unexpected and seen with people such as for example Holtsberg, when the PEG of different molecular weight opposite with the result of arginine deaminase (from microbe-derived isolating another amino acid degradation enzyme) when puting together.In those researchs; The pharmacokinetics of arginine deaminase and pharmacodynamic feature are along with the size of PEG attachment is that 5000Da increases to 20 from molecular weight; 000Da and increase (Holtsberg, F.W., Journal of Controlled Release 80 (2002); 259-271), all incorporate into by reference with it at this).

Table 5

In addition; As hereinafter see in more detail; The immunogenicity data show goes out 10kDa-100% and has shown unacceptable immunogenicity characteristic spectrum; When considering compound administration to irritated to the bacillus coli L-asparaginase enzyme or when having produced the patient of anti--altheine enzyme antibody, this is main shortcoming.This respect, 10kDa-100% conjugate are really inappropriate.2kDa-100% and 5kDa-100% are preferred, and the 5kDa-100% conjugate is preferred especially.

Embodiment 10: immunogenicity

In B6D2F1-heterozygote (immunocompetent, female) (Charles River Germany), measured the immunogenicity of mPEG-r-Ke Lita enzyme conjugate.In 1,2,3,4 and 8 weeks, biweekly (handle animal for

and 5U/kg bw (for all r-Ke Lita enzyme conjugates) through i.v. injection 250U/kg bw.1h and 1w before administration, 2w, 4w behind 6w and the 8w, collects blood serum sample from the eye hole.Confirm in the serum level of anti--Ke Lita enzyme or anti--mPEG-r-Ke Lita enzyme antibody through ELISA.The result is summarised in Figure 10 and 11.

Since observing high anti-Ke Lita enzyme antibody titre the 2nd week for

and its quilt in whole conceptual phase is kept.On the contrary, do not observe significant antibody horizontal (Figure 10) for r-Ke Lita enzyme conjugate.

As shown in Figure 11, anti-conjugate production of antibodies keeps low-intensity and frequency for 2kDa and 5kDa mPEG-r-Ke Lita enzyme conjugate, and for 10kDa mPEG-r-Ke Lita enzyme conjugate, it increases with higher value and frequency.Do not observe tangible difference (not shown) between the conjugate for complete and part Pegylation.

Therefore, these data show, and compare without the altheine enzyme of modifying, and selected Pegylation strategy has reduced the immunogenicity of conjugate, has significantly reduced anti-Ke Lita enzyme antibody and has replied.Yet, detected anti-conjugate antibody, when particularly using the 10kDa conjugate, lower intensity is arranged during with 2kDa and 5kDa conjugate.

In a word; As if until 5kDa; Pegylation is being successful aspect the pharmacokinetics characteristic spectrum, effectiveness and the acting duration that improve r-Ke Lita enzyme; And reduced immunogenicity with comparing without the albumen of modifying, increased along with the size of employed polymer and render a service and acting duration, 5kDa mPEG-r-Ke Lita enzyme is puted together more effective slightly than 2kDa mPEG-r-Ke Lita enzyme conjugate.Yet further the size with PEG is increased to 10kDa not further improvement effectiveness and acting duration, because 10kDa mPEG-r-Ke Lita enzyme conjugate does not have 5kDa mPEG-r-Ke Lita enzyme conjugate effective in vivo, though vitro efficacy is similar.In addition, 10kDa mPEG-r-Ke Lita enzyme conjugate has shown unacceptable immunogenicity characteristic spectrum, and in view of other the proteic result who has delivered, this is beat all result.

Though through explaination and the mode of embodiment, on some details, described embodiment of the present invention and application, those skilled in the art obvious are: possibly carry out many other modification and inventive concepts of not departing among this paper to be contained.All lists of references of quoting in this article all are merged in it.

Claims

1. conjugate; It comprises from altheine enzyme of Erwinia (Erwinia) and Polyethylene Glycol (PEG); The aminoacid of said altheine enzyme and SEQ ID NO:1 has at least 80% homogeneity, and wherein the molecular weight that has of PEG is less than or equal to about 5000Da.

2. the conjugate of claim 1, the aminoacid of wherein said altheine enzyme and SEQ ID NO:1 has at least 90% homogeneity.

3. the conjugate of claim 1, the aminoacid of wherein said altheine enzyme and SEQ ID NO:1 has at least 99% homogeneity.

4. the conjugate of claim 1, wherein said altheine enzyme is identical with the aminoacid of SEQ ID NO:1.

5. each conjugate among the claim 1-4, wherein said PEG has the molecular weight of about 5000Da.

6. each conjugate among the claim 1-4, the molecular weight that wherein said PEG has is less than about 5000Da.

7. each conjugate among the claim 1-4, the molecular weight that wherein said PEG has is less than about 4000Da.

8. each conjugate among the claim 1-4, the molecular weight that wherein said PEG has is less than about 3000Da.

9. each conjugate among the claim 1-4, the molecular weight that wherein said PEG has is less than about 2500Da.

10. each conjugate among the claim 1-9, wherein the altheine enzyme when not puting together with PEG is compared, and the external activity that said conjugate has is at least 60%.

11. the conjugate of claim 10, wherein the altheine enzyme when not puting together with PEG is compared, and the external activity that said conjugate has is at least 70%.

12. the conjugate of claim 11, wherein the altheine enzyme when not puting together with PEG is compared, and the external activity that said conjugate has is at least 78%.

13. the conjugate of claim 10, wherein the altheine enzyme when not puting together with PEG is compared, and the external activity that said conjugate has is at least 80%.

14. the conjugate of claim 10, wherein the altheine enzyme when not puting together with PEG is compared, and the external activity that said conjugate has is at least 87%.

15. the conjugate of claim 10, wherein the altheine enzyme when not puting together with PEG is compared, and the external activity that said conjugate has is at least 90%.

16. each conjugate among the claim 1-15, wherein the altheine enzyme when not puting together with PEG is compared, and the altheine that said conjugate has consumes active more effective at least about 50 times.

17. each conjugate among the claim 1-16, wherein said conjugate consume blood plasma altheine level to detect less than level continue at least 48 hours.

18. each conjugate among the claim 1-16, wherein said conjugate consume blood plasma altheine level to detect less than level continue at least 96 hours.

19. each conjugate among the claim 1-18, wherein the altheine enzyme when not puting together with PEG is compared, and said conjugate has the longer body-internal-circulation half-life.

20. each conjugate among the claim 1-19, wherein under the albumen dosage that equates, said conjugate has the t longer than pegaspargase _1/2

21. the conjugate of claim 20, wherein under the dosage of about 50 μ g albumen/kg, said conjugate has the t at least about 58 hours _1/2

22. the conjugate of claim 20, wherein under the dosage of about 50 μ g albumen/kg, said conjugate has the t at least about 60 hours _1/2

23. the conjugate of claim 20, wherein under the dosage of about 50 μ g albumen/kg, said conjugate has the t at least about 63 hours _1/2

24. the conjugate of claim 20, wherein under the dosage of about 50 μ g albumen/kg, said conjugate has the t at least about 65 hours _1/2

25. the conjugate of claim 20, wherein under the dosage of about 10 μ g albumen/kg, said conjugate has the t at least about 34 hours _1/2

26. the conjugate of claim 20, wherein under the dosage of about 10 μ g albumen/kg, said conjugate has the t at least about 36 hours _1/2

27. the conjugate of claim 20, wherein under the dosage of about 10 μ g albumen/kg, said conjugate has the t at least about 38 hours _1/2

28. the conjugate of claim 20, wherein under the dosage of about 10 μ g albumen/kg, said conjugate has the t at least about 40 hours _1/2

29. each conjugate among the claim 1-28, wherein the altheine enzyme when not puting together with PEG is compared, and said conjugate has bigger AUC.

30. the conjugate of claim 29, wherein under the albumen dosage that equates, the average A UC that said conjugate has is bigger at least about 3 times than pegaspargase.

31. each conjugate among the claim 1-30, wherein PEG is covalently attached to one or more amino of said altheine enzyme.

32. the conjugate of claim 31, wherein PEG is covalently attached to said one or more amino through amido link.

33. the conjugate of claim 31 or 32, wherein PEG is covalently attached to the available amino end at least about 40% to about 100%.

34. the conjugate of claim 31, wherein PEG is covalently attached to the total amino at least about 40% to about 90%.

35. the conjugate of one of claim 1 to 34, it has following formula:

Asp-[NH-CO-(CH2)x-CO-NH-PEG]n

Wherein Asp is the altheine enzyme; NH is lysine residue and/or the terminal one or more NH groups of N-among the Asp; PEG is a polyalkylene glycol moiety; N is a numeral of representing among the Asp at least 40% to about 100% available amino end (for example lysine residue and/or N-are terminal), and x is the integer in 1 to 8 scope.

36. the conjugate of claim 35, wherein x is the integer in 2 to 5 scopes.

37. the conjugate of claim 35, wherein n is the numeral of representing among the Asp at least about 40% available amino end.

38. the conjugate of claim 37, wherein n is a numeral of representing among the Asp about 100% available amino end.

39. each conjugate among the claim 1-38, wherein said PEG are mono methoxy polyethylene glycol.

40. be used for preparing each the method for conjugate of claim 1-39, said method comprises makes a certain amount of said PEG in solutions buffered, combine to be enough to make covalently bound a period of time to said altheine enzyme of said PEG with a certain amount of said altheine enzyme.

41. the method for claim 40, wherein said have a pH value of about 7.0 to about 9.0 through solutions buffered.

42. the method for claim 41, wherein said have a pH value of about 7.5 to about 8.5 through solutions buffered.

43. the method for claim 40, the amount of said altheine enzyme are the protein concentration of about 0.5mg/mL to about 25mg/mL.

44. the method for claim 43, the amount of wherein said altheine enzyme are the protein concentration of about 2mg/mL to about 20mg/mL.

45. the method for claim 43, the amount of wherein said altheine enzyme are the protein concentration of about 3mg/mL to about 15mg/mL.

46. the method for claim 40, the amount of wherein said PEG is: polymer is less than about 20: 1 than the molar excess of the amino in the said altheine enzyme.

47. the method for claim 46, the amount of wherein said PEG is: polymer is less than about 10: 1 than the molar excess of the amino in the said altheine enzyme.

48. being polymer, the method for claim 46, the amount of wherein said PEG be less than about 8: 1 than the molar excess of the amino in the said altheine enzyme.

49. each method among the claim 40-48, wherein said PEG is a mono methoxy polyethylene glycol.

50. the method for the disease that can treat through altheine consumption among the treatment patient, said method comprise the conjugate of using among the claim 1-39 of effective dose each to said patient.

51. the method for claim 50, wherein said can be cancer through the disease that altheine consumption is treated.

52. the method for claim 51, wherein said cancer is selected from: acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, NK lymphoma and cancer of pancreas.

53. the method for claim 52, wherein said cancer are ALL.

54. the method for claim 53 is wherein used said conjugate with about 5U/kg to the amount of about 25U/kg.

55. the method for claim 54 is wherein used said conjugate with the amount of about 25U/kg.

56. each method among the claim 53-55 is wherein used said conjugate with following dosage: its consume altheine to detect less than level continue about 3 days to about 10 days.

57. each method among the claim 53-56 is wherein compared with unconjugated altheine enzyme, said conjugate causes lower immunogenic response in said patient.

58. each method among the claim 53-57 is wherein compared with unconjugated altheine enzyme, said conjugate has the longer body-internal-circulation half-life behind single dose.

59. each method among the claim 53-58 is wherein compared with unconjugated altheine enzyme, said conjugate has bigger AUC value behind single dose.

60. each method among the claim 50-59, wherein said conjugate is by intravenous administration.

61. each method among the claim 50-59, wherein said conjugate is used by intramuscular.

62. each method among the claim 50-61 is wherein used once said conjugate weekly.

63. each method among the claim 50-61 is wherein used said conjugate weekly twice.

64. each method among the claim 50-61 wherein is less than and uses said conjugate once in a week.

65. each method among the claim 50-64, wherein said conjugate is used as monotherapy.

66. the method for claim 65, wherein said conjugate is not used with the asparagine synthetase inhibitor.

67. each method in the claim 50 to 66, wherein said patient has hypersensitivity previously to bacillus coli L-asparaginase enzyme or its Pegylation form.

68. each method in the claim 50 to 66, wherein said patient has hypersensitivity previously to Erwinia altheine enzyme.

69. the method for claim 67 or 68, wherein said hypersensitivity are selected from anaphylaxis, anaphylactic shock and reticent hypersensitivity.

70. each method among the claim 50-69, wherein said patient has palindromia.

71. the method for claim 70, wherein said palindromia takes place after the form of therapy with bacillus coli L-asparaginase enzyme or its Pegylation.

72. pharmaceutical composition, it comprises among the claim 1-39 each conjugate.

73. each conjugate among the claim 1-39, it is used for treatment.

74. the conjugate of claim 73, it is used to treat the disease that can treat through altheine consumption.

75. the conjugate of claim 74, wherein said disease are cancer.

76. the conjugate of claim 75, wherein said cancer are ALL.

77. the conjugate of claim 76 is wherein compared with unconjugated altheine enzyme, said conjugate causes lower immunogenic response in said patient.

78. each conjugate among the claim 73-77, wherein said medicine are used for treating bacillus coli L-asparaginase enzyme or its Pegylation form had the patient's of hypersensitivity previously disease.

79. each conjugate among the claim 73-77, wherein said medicine are used for treating the disease that Erwinia altheine enzyme is had the patient of hypersensitivity previously.

80. the conjugate of claim 78 or 79, wherein said hypersensitivity are selected from anaphylaxis, anaphylactic shock and reticent hypersensitivity.

81. each conjugate among the claim 73-80, wherein said medicine is used for treating the patient's with palindromia disease.

82. the conjugate of claim 81, wherein said palindromia take place after with bacillus coli L-asparaginase enzyme or its Pegylation form of therapy.

83. the method for the disease that can treat through altheine consumption among the treatment patient; Said method comprises patient that selection need be treated and uses among the claim 1-39 of effective dose each conjugate to said patient; Wherein said patient has hypersensitivity previously to bacillus coli L-asparaginase enzyme or its Pegylation form; And/or wherein said patient has hypersensitivity previously to Erwinia altheine enzyme, and/or said patient has palindromia.

84. the method for claim 83, wherein said hypersensitivity are selected from anaphylaxis, anaphylactic shock and reticent hypersensitivity.

85. the method for claim 83, wherein said palindromia take place after with bacillus coli L-asparaginase enzyme or its Pegylation form of therapy.

86. the method for one of claim 83-85, wherein said can be cancer through the disease of altheine consumption treatment, particularly is selected from acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, NK lymphoma and cancer of pancreas.

87. the method for one of claim 83-86, wherein with about 5U/kg to the amount of about 25U/kg, preferably the amount of about 25U/kg is used said conjugate.

88. each method among the claim 83-87 is wherein used said conjugate with following dosage: its consume altheine to detect less than level continue about 3 days to about 10 days.

89. each method among the claim 83-88 is wherein used said conjugate intravenous ground or intramuscular.

90. each method in the claim 83 to 89 is wherein used said conjugate once in a week.

91. each method among the claim 83-89, the wherein said conjugate of administered twice weekly.

92. each method among the claim 83-89 wherein is less than and uses said conjugate once in a week.

93. each method among the claim 83-92, wherein said conjugate is used as monotherapy.

94. the method for claim 93, wherein said conjugate is not used with the asparagine synthetase inhibitor.